INTRODUCTION:

Natural product remain the most prolific source of new antimicrobials, as well as its chemical diversity is still unmatched by combinatorial chemistry efforts¹. Actinomycetes is widely accepted as an applicable source of antimicrobial compounds production. The marine Actinomycetes can be stated as a source of novel antimicrobial compounds with much higher pharmaceutical values as many pathogenic bacteria develop resistance against commercially available antibiotics which indicates the failure of terrestrial microorganisms as a source of potential antimicrobial compounds^2-3. Several research works have been already performed and it was found that marine Actinomycetes contribution as an alternative source of biologically active substances is unpredictable^4-6. At present two third of the naturally occurring potent antibiotics are derived from marine Actinomycetes⁷.

Marine Actinomycetes are characterized as gram positive aerobic mycelial prokaryotes with high G+C content⁸. Among the Actinomycetes group one of the genus called Nocardiopsis is assumed as a promising source of new and potent antimicrobial compounds⁹.Mutation is a method to activate antibiotic production in marine Actinomycetes by enhancing the activity of particular gene responsible for its antimicrobial activity¹⁰.A number of work has been done on this concept of dramatic activation of antibiotic production¹¹.The power of this approach can be elegantly exemplified by the work on strain improvement for the yield of antibiotic. However, a molecular basis of data is needed before confirming the phenomenon of mutation which can be best achieved by a technique known as RAPD (Random Amplification of Polymorphic DNA). RAPD is a Polymerase Chain Reaction (PCR) based method which can be used to distinguish between strains within a species¹². Along with mutation, among the various methods for enhancing the activity of antibiotic production, one of the most reliable and effective technique is optimization of production media. Since the provided normal condition along with the synthetic media fails to operate and becomes uneconomical for the production of various metabolites in high concentration. Therefore, it is important to optimize the conditions of medium for the growth of organism which will ultimately increase the production yield otherwise the cost of the medium along with other commodities can affect the overall economics¹¹. Optimization refers to designing a fully perfect, effective system that should involve mathematical techniques since statistical optimization allows effective screening of a huge experiment subunits and it also considers the significance of each components¹³. To optimize the condition, statistical optimization which is commonly known as Response Surface Methodology (RSM) is widely used¹⁴. RSM is a collection of statistical techniques for constructing experiments, building models and evaluating the effect of factors and to screen optimum conditions of factors for desirable responses which allows its interactions along with the role of each component¹⁵. It is an industrially efficient tool for the production of commercially important drugs¹⁴. Nosocomial infections occur within 48 hours of hospital admission or 3 days of discharge or 30 days post operation¹⁶. P. aeruginosa is one of the causative organisms for the infections. It poses a therapeutic problem as it is a multidrug resistant organism. It is resistant against Penicillins, Cephalosporins, Quinolones, Monobactums, Imipenems and Aminoglycosides¹⁷. Developing an appropriate media and various conditions are of great importance to enhance the efficiency as well as productivity of bioactive metabolites process as it can significantly affects the product concentration and the yield. There are reports where RSM have been employed in the study to enhance antibacterial activity of Streptomyces sp. However to optimize a medium for the production of high amount of antibiotic using RSM for mutant Nocardiopsis sp are not available. Therefore, the present study explains the optimization of the production media with the effect of mutation for strain improvement for the production of antibiotic in Nocardiopsis VITSRTB.

MATERIALS AND METHODS:

Media:

All the media were purchased from Hi media Pvt. Ltd, Mumbai and chemicals from Sisco Research Laboratories (SRL) Mumbai.

Isolation of marine Actinobacteria:

Marine water samples were collected from Marina beach, Chennai (13.0542°N, 80.2837° E). The samples were serially diluted, filtered and sterilized with 50% marine water to the dilution of 10^-7. Each 100 µl of serially diluted samples were poured in Kuster’s Agar plates (pH -7.2) prepared in 50% seawater to maintain the growth of Actinobacteria¹⁸. To prevent the growth of bacterial and fungal contamination cycloheximide (100 mg/l) and nalidixic acid (20 mg/l) was added respectively¹⁹. The plates were incubated at 28 ± 2º C and colonies were observed from 5^th day onwards to 1 month.^[18] The Actinobacteria strains were purified by continuous streaking on Yeast Extract- Malt Extract agar (ISP 2) and stored at 4 ± 2º C.

Test organism:

The clinical isolate of Staphylococcus aureus, Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa and Bacillus subtilis, were obtained from Narayani hospital, Vellore district, Tamil Nadu, India.

Positive control:

Nalidixic acid (30 μg/disc) was used as positive control for S. typhi, Gentamicin (10 μg/disc) for P. aeruginosa, Methicilin (5µg/disc) for S. aureus and Erythromycin (10 μg/disc) for B. subtilis.

Antibiogram Assay:

Antibiogram assay was carried out against P.aeruginosa by using commercially available antibiotic discs (Hi Media Pvt. Ltd. Mumbai, India) of Erythromycin, Amikacin, Neomycin, Ceftazidime, and Gentamycin²⁰.

Screening for antibacterial activity by Agar well diffusion method:

The marine Actinobacteria isolates were inoculated in SS Media broth and then incubated for 7 days at room temperature. After 7 days, broths were centrifuged at 8500 rpm for 15 mins. The supernatant were collected. Muller Hinton agar plates were prepared and lawn culture made using sterile cotton swabs of the test pathogen. Wells were made on the agar plates by the help of sterile cork borer and 100 µl of collected supernatant was poured on the wells using sterile micro-tips²¹. The plates were then incubated for 24 hours at room temperature. The zone of inhibition was observed on the plates after 24 hours of incubation.

Phenotypic characterization of potential strain

1. Spore chain morphology:

The strains were grouped on the basis of spore chain morphology into different sections namely rectiflexibils (RF), retinaculiaperti (RA) and spiral (S). Characterization of spore chains was determined by direct microscopic inspection of culture surface. Spore morphology characters of these strains were observed by inoculating a loopful of 7 days old culture in to 1.5% agar medium contained in test tube at 37º C. The actinomycetes were suspended and mixed in the semisolid agar medium and 2 drops of the medium were aseptically pipette on to a sterile glass slide. A thin film of agar was made by spreading a drop of agar on the slide to facilitate direct examination under microscope¹⁸.

2. Molecular characterization:

The molecular characterization of the potent strain showing significant antimicrobial activity was carried out with 16S rRNA gene sequencing. The Actinobacterial DNA was isolated by using HiPurA bacterial DNA isolation and purification kit (Himedia pvt. ltd, India) and amplification was done by PCR technique, using a master mix kit, Medoxmix (Medoxpvt. ltd, India) as per user manual. The forward and reverse primers used are 5'- AGA GTY TGA TC(AC) TGN CTY AG-3' and 5'-TAC GG(CT) TAY CTT GTN ACR ACY T-3'respectively. The similarity and homology of the16S rRNA partial gene sequence was analyzed with the similar existing sequences available in the data bank of National Centre for Biotechnology Information (NCBI) using BLAST search. The DNA sequences were aligned and phylogenetic tree was constructed through neighbour joining method viz., ClustalW software²².

Induction of Mutations in potential isolate:

The potential isolate was mutated by chemical mutagen in order to check the frequency of mutation.

Chemical mutation:

The marine Nocardiopsis VITSRTB (HF546396) strain was cultured in Actinomycetes Isolation Agar (AIA) plates containing 10 ppm of sodium azide²³, and incubated at room temperature for 7 days. After incubation, the effect of mutation was observed for antibacterial activity against P. aeruginosa.

DNA extraction and PCR amplification:

The CTAB (hexadecyltri-methylammonium bromide) method²⁴, was used to extract total DNA from the wild and mutated strain of Nocardiopsis VITSRTB (HF546396). Bacterial pellet 100 mg was taken and suspended with 2 ml of extraction buffer [1.4 M NaCl, 100 mM Tris-HCl (pH 8.0), 20 mM EDTA, 2% CTAB, and 1% 2- mercaptoethanol] at 65°C, and incubated at 65°C for 45 mins with slow shaking for every 10 mins. Protein was extracted by using twice the volume of 2 ml chloroform: iso-amylalcohol (24:1) and centrifuged at 13,000 rpm for 2 mins. RNase (10 µg/ml) was added to the supernatants for 30 mins at 37°C and the mixture was again centrifuged at 13,000 rpm for 2 mins. Amplification of genomic DNA was made on an Agilent cycler 2200 (Germany), using the arbitrary primers (decamers). The 6 primers were purchased from Amnion Biotech, Bangalore (Table 1); the six different 10-mer oligonucleotide RAPD primers used in this study were OPA-1, OPA-2, OPA-3, OPA-4, OPA-5 and OPA-7 (Table 1). Amplification of genomic DNA was performed in 25-µl reaction volumes containing 1.2 units of Taq polymerase (Sangon, Shanghai, China), 10 mM Tris-HCl (pH 9.0), 25 mM KCl, 2 mM MgCl2 , 0.2 mM of each dNTP, 24 ng each of random primer and 40 ng of template DNA. The cycle program included an initial 75 secs denaturation at 94°C, followed by 45 cycles of 15 secs at 94°C, 30 secs at 42°C and 75 secs at 72°C, with a final extension at 72°C for 7 mins. RAPD fragments were separated electrophoretically on 1.5% agarose gels in 1X TBE buffer, stained with ethidium bromide and photographed on a UV transilluminator using a digital camera. DNA was amplified with the same primer more than once and the banding patterns were compared.

Optimization of Media by RSM:

1. Selection of nitrogen and carbon sources:

Various nitrogen sources (peptone, beef extract, yeast extract and casein), polysaccharide (starch, cellulose, sucrose, lactose and maltose) and monosaccharide (glucose, xylose, dextrose and fructose) sources were used as substitutes for nitrogen and carbon sources of the basic media²⁵.

Table 1. Arbitrary 10-mer primers used in RAPD analysis

S.No	Primer name	Sequence
1	OPA-01	CAGGCCCTTC
2	OPA-02	TGCCGAGCTG
3	OPA-03	AGTCAGCCAC
4	OPA-04	AATCGGGCTG
5	OPA-05	AGGGGTCTTG
6	OPA-07	GAAACGGGTG

2. Experimental setup:

Based on the selected nitrogen and carbon sources, the broths were prepared and inoculated with both wild and mutated strain of Nocardiopsis VITSRTB and incubated for seven days at room temperature. After the incubation period, the broths were centrifuged and the supernatant was collected to perform antibiotic assay against P. aeruginosa. Based on the results, the five variables (cellulose, fructose, casein, temperature, and pH) were considered and RSM was carried out by using Design Expert, version 7.0 (Table 2).

Table 2. Variables considered for RSM with lower and upper limit

Variables	Variable code	Lower limit	Upper limit
Cellulose	A	22	30
Fructose	B	8	15
Casein	C	1	5
Temperature	D	28	37
pH	E	6.5	8.5

Isolation of biologically active compounds was done by solvent extraction method. The Nocardiopsis VITSRTB inoculated broth was centrifuged at 8500 rpm for 15 minutes at 4°C and the supernatant was mixed with a mid-polar solvent such as Ethyl acetate and shaken vigorously for one hour in a separating funnel. The mixture of the solvent and filtrate were left for 24 hrs for proper stabilization. After 24 hrs, the solvent phase was separated from aqueous phase. The extracted solvent phase was concentrated and used for examination antibacterial activity.

GC-MS analysis:

For the identification of bioactive compound secreted by the potent strain Gas-chromatography and Mass-spectrophotometry were performed using (Aligent Technologies United State, GC-MS) assembly equipped with HP-5 cross linked fused silica capillary column (25 m \ 0:32 mm 0.25 µm). Helium (99.999%) was used as capillary gas at 38 cm/s. Flow rate was maintained as 1ml/min. In normal temperature conditions, split less injector at 280º C and column temperature program of 80º C CD 310º C at 10º C /min was maintained. Mass detection from 50 Da to 700 Da was analysed. Sample was dissolved in ethyl acetate (1:1 v/v) at room temperature for 24 hrs before analysis.

RESULTS AND DISCUSSION:

Isolation of actinobacteria:

A total of 10 marine Actinobacteria isolates were isolated from marine water sample. Actinobacteria colonies were selected based on colony morphology and microscopic appearance. The isolates were designated as SRTB1-SRTB10.

Antibiogram Assay:

Erythromycin was found to be most effective against P.aeruginosa (24 ± 2.08) mm where as Ceftazidime showed a zone of inhibition of (14 ± 1.52) mm (Table 3).

Table 3. Antibiogram Assay for P. aeruginosa

Antibiotics	Zone of Inhibition in mm
Erythromycin	24 ± 2.08
Amikacin	20 ± 3.78
Neomycin	20 ± 2.51
Ceftazidime	14 ± 1.52
Gentamycin	18 ± 1.82

Antibacterial activity of isolated actinobacteria:

Actinobacteria isolates were screened for antibacterial activity against four clinical pathogens S.aureus, B.subtilus, E.coli,P.aeruginosa and S. typhi. Among 10 isolates, 3 isolates of actinobacteria (SRTB3, SRTB4 and SRTB5) showed good activity towardstest pathogensin agar well diffusion method. The maximum zone of inhibition was shown by SRTB3 (12 ± 0.40 mm), SRTB4 (10 ± 0.34 mm) and SRTB5 (8 ± 0.45 mm) against P. aeruginosaas summarized in Table 4.

Phenotypic and molecular characterization:

Phenotypic characterization showed the aerial mycelium pigments and reverse side pigments which were tabulated in Table 5. The spore chain morphology of the potent isolate was observed to be showing similar structures as present inNocardiopsis sp.The BLAST search of 16S rRNA sequence of the strain showed highest similarity (98%) with Nocardiopsis and phylogenetic tree was constructed by using tree view software (Fig. 1). Based on the molecular taxonomy and phylogeny the strain was identified as Nocardiopsiswhich now designated as NocardiopsisVITSRTB. The nucleotide sequence data obtained in the study was deposited in NCBI under accession number (HF546396). A neighbour-joining tree based on 16S rRNA sequences showed that the isolate occupies a distinct phylogenetic position within the representatives of the Nocardiaceaefamily. The 16S rRNA sequence is highly conserved sequence. The 16S rRNA genes have provided valuable data for constructing phylogenies at and above the genus level²⁶.

Effect of mutation on antimicrobial activity of marine actinobacteria:

Out of the 10 Actinobacteria strain, the strain SRTB3 has shown maximum antimicrobial activity (12 ± 0.40) against P. aeruginosa and was identified as Nocardiopsis VITSRTB (HF546396). The strain NocardiopsisVITSRTB (HF546396) was then subjected to chemical mutation. The chemically mutated strain, showed effective potential (21± 0.57 mm) Fig. 2 (B) against P. aeruginosa as compare to the wild strain of Nocardiopsis VITSRTB (HF546396) as shown in Fig. 2 (A). The mutated strain was found to show increase in antibacterial activity and significant increase in zone of inhibition. Likewise, Phillips²⁷, reported that UV mutated Penicillumchrysogenum produced antibiotics 10,000 times greater than that of nonmutated strains. Similarly, Muthurayer et al²⁸. reported that UV mutated Actinomycetes strains AA-13 showed increase in antimicrobial activity (+12 mm) towards human bacterial pathogen (K. pneumoniae). In our study the chemically mutated strain showed significant increase in antibacterial activity. This is due to the chemical mutation which enhance the transcription and translation of secondary metabolite genes in stationary phase. The mutagenic effect of sodium azide on actinomycetes has also been reported by Bhattarai et al²⁹. Thus, chemical mutagens can be used for the strain improvement. Strain improvement is widely allowed for the selection of particular strain which is having antimicrobial activity. This is mainly due to the phenomenon of alteration in gene regulation which leads to the enhancement of the production of antibiotics³⁰.

Fig. 2 Antibacterial activity of NocardiopsisVITSRTB (A) wild strain and (B) chemically mutated strain

RAPD analysis:

RAPD fingerprints profiles were generated using six primers. The images of agarose gel separation of RAPD for wild and the chemically mutated strains are shown in Fig. 3. This suggests that the frequency of mutation was much higher in chemically mutated strain as compare to the wild strain. Thus chemically mutated strain was further optimized by using RSM to increase the antibacterial activity.

Fig. 3 Image of agarose gel separation for (A) wild strain and (B) Chemically mutated strain of Nocardiopsis VITSRTB. The L-1- Primer-1, L-2- Primer-2, L-3- Primer-3, L-4 I Kb DNA ladder, L-5- Primer-4, L-6- Primer-5, L-7- Primer-6

RSM analysis:

The quadratic model gave the following equation for the wild strain:

R1 = + 4.54 + 0.81 * A + 1.75* B + 0.18 * C - 0.47 * D - 3.31* E - 0.080 * A * B + 0.69* A * C - 1.42 * A * D - 0.088 * A * E - 0.038 * B * C - 3.87 * B * D + 0.41 * B * E + 0.69 * C * D - 0.16 * C * E + 0.13 * D * E - 1.29 * A2 - 1.73 * B2 - 7.59 * C2 + 1.73 * D2 + 9.74 * E2

The quadratic model gave the following equation for the chemically mutated strain:

R1 = + 5.83 + 2.33 * A + 3.76 * B +0.78 * C + 1.62 * D - 3.68 * E +0.080 * A * B + 0.35 * A * C-0.71 * A * D + 2.00 * A * E - 2.12 * B * C - 4.06 * B * D + 0.42 * B * E + 1.94 * C * D - 1.32 * C * E - 1.04 * D * E - 0.90 * A2 - 7.01 * B2 - 11.26 * C2 + 6.29 * D2 +16.66 * E2

The F value is determined based on the F-test. F-test measures the ratio of the variance between groups to the variance within groups³¹. The model for both wild and mutated strain was seen to be significant and this shows that the F value has to be greater than the standard table value. The F value for both wild and mutated strain was seen to be significant because it is higher than 0.05. An F value greater than 0.05 is considered and a value less than 0.100 is considered as insignificant³². The p-value or probability value that points out which effects can be considered significant, was seen to be less than 0.01 and such a low value indicates that there is a statistically significant relationship between the variable within a 99% confidence interval. The p-value indicates a non-significant relative to pure error.

The co-efficient of determination, R², for antibacterial activity was calculated as 0.8831 and 0.8789 for wild and mutated strain respectively. The closer the value of R²to unity, the better the empirical models fits the experimental data³³. The smaller the value of R², the lesser will be the relevance of the dependant variables in the model has in explaining the behaviour of variations. This indicated that the statistical model explained 88.31% and 87.89% of correlation between the predicted and observed values. Since the R² value in the case of wild and mutated has shown to be closer to 1, there is a good agreement between the experimental and predicted values of antibacterial activity. Fig. 4 shows the normal plot of residues for the wild and mutated strain of NocardiopsisVITSRTB and their 3D interaction between variables is shown in Fig. 5. The zone of inhibition after mutation was observed as 21 mm. However, after optimization of the media, the zone of inhibition increased to 32 mm. Therefore it can be seen that optimization of the media has also increased the productivity of the antimicrobial agent. The model for both wild and mutated strain was seen to be significant and this shows that the F value has to be greater than the standard table value. The F value for both wild and mutated strain was seen to be significant because it is higher than 0.05. An F value greater than 0.05 is considered and a value less than 0.100 is considered as insignificant.P-value or probability value less than 0.01 indicates that there exists a statistically significant relationship between the variable within a 99% confidence interval. The p-value indicates a non-significant model relative to pure error. The value of R² was seen to be almost near 1.The closer the value of R²to unity, the better the empirical models fits the experimental data. The smaller the value of R², the lesser will be the relevance of the dependant variables in the model has in explaining the behaviour of variations³⁴. This indicated that the statistical model explained 88.31% and 87.89% of correlation between the predicted and observed values. Since the R² value in the case of wild and mutated has shown to be closer to 1, there is a good agreement between the experimental and predicted values of antibacterial activity.

Fig. 4 The normal plot for (A) wild and (B) chemically mutated strain of Nocardiopsis VITSRTB

Fig. 5 3D interactions between substrates for (A) wild strain where (I) fructose and cellulose, (II) casein and cellulose, (III) temperature and cellulose, (IV) pH and cellulose (V) casein and fructose (VI) temperature and fructose (VII) pH and fructose (VIII) temperature and casein (IX) pH and casein and (X) pH and temperature on the antbacterial activity for the wild strain and (B) chemically mutated strain where (I) temperature and fructose, (II) pH and fructose, (III) temperature and casein, (IV) pH and casein, (V) pH and temperature, (VI) pH and fructose, (VII) temperature and fructose, (VIII) temperature and casein, (IX) pH and casein and (X) pH and temperature on the antimicrobial activity for the mutated strain.

GC-MS Analysis

The components present in the ethyl acetate extract of wild and chemically mutated strain of Nocardiopsis VITSRTB were identified by GC-MS. The chromatogram is shown in (Fig. 6). Four components were identified in the ethyl acetate extract of wild strain of Nocardiopsis sp. The compounds were Ethyl 1,4-methyl hexadecanoate, Phenol 2,4 bis (1,1dimethyl-), Ethyl heptadecanoate and Phenol 3,5 bis (1,1dimethyl-). Twelve components were identified in the ethyl acetate extract of mutated strain of Nocardiopsis VITSRTB 4,5 Octanediol is one of the major component in the extract. The compound, Ethyl 1,4-methyl hexadecanoate, is a derivative of decanoic acid and it has been found that hexadecanoic acid has an antimicrobial activity³⁵. Hence, it can be stated that the compound, Ethyl 1,4-methyl hexadecanoate, has an antimicrobial activity. Similarly, The compound,Octanediol is a derivative of 1,8 Octanediol, and it shows bactericidal as well as bacteriostatic activity³⁵. Hence, it can be stated that the compound, 4,5 Octanediol, has an antibacterial activity.

Fig. 6 GC-MS chromatogram of ethyl acetate extract of (A) Wild strain of Nocardiopsis VITSRTB and (B) Chemically Mutated strain of Nocardiopsis VITSRTB

CONCLUSIONS:

The present study concluded that the potential Actinobacteria strains with antibacterial activity were isolated. The strain having the maximum antibacterial activity was mutated and the production media was optimized by RSM. The antibacterial activity was tested against the clinical isolate of P. aeruginosa. A three-fold increase in the activity was observed when the strains were mutated and optimized. Hence, it can be concluded that the combination of the two methods, i.e. mutation and optimization, is an effective method for increasing the efficacy of antibacterial activity of both wild and mutated strain of NocardiopsisVITSRTB. Moreover, The bioactive compounds were found to be ethyl 1,4- methyl hexadecanoate and 4,5- Octanediol in wild and chemically mutated strain of Nocardiopsis VITSRTB respectively are found to be having bactericidal, antimicrobial and bacteriostatic activity. Hence, it can be assumed that this two major compounds plays a significant role for the antibacterial activity of marine Nocardiopsis VITSRTB. However, further studies are needed to characterize the pure compound.

ACKNOWLEDGEMENT:

The authors are very much grateful to the management and staff of VIT University, Tamil Nadu, India for supporting this study.

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Received on 11.12.2013 Modified on 04.01.2014

Research J. Pharm. and Tech. 7(2): Feb. 2014; Page 213-220

Stain no.	S.aureus	B.subtilis	E.coli	P.aeruginosa	S. typhi
SRTB-1	2 ± 0.40	3 ± 0.37	5 ± 0.35	6 ± 0.41	2 ± 0.40
SRTB-2	4 ± 0.41	3 ± 0.37	--	4 ± 0.41	--
SRTB-3	5 ± 0.35	_	5 ± 0.35	12± 0.40	1± 0.11
SRTB-4	4 ± 0.41	--	3 ± 0.37	10 ± 0.34	--
SRTB-5	3 ± 0.37	6 ± 0.41	4 ± 0.41	8 ± 0.45	--
SRTB-6	3 ± 0.37	6 ± 0.41	5 ± 0.35	3 ± 0.37	--
SRTB-7	2 ± 0.40	4 ± 0.41	5 ± 0.35	6 ± 0.41	--
SRTB-8	--	--	--	--	--
SRTB-9	3 ± 0.37	6 ± 0.41	_	5 ± 0.35	--
SRTB-10	3 ± 0.37	3 ± 0.37	3 ± 0.37	--	--

Medium	Strain
Medium	Aerial mycelium pigment	Reverse side pigment
ISP 1	White	Yellow
ISP 2	Dark yellow	White
ISP 3	White	White
ISP 4	sea green	White
ISP 5	Brownn	White
ISP 6	Sky	White
ISP 7	White	White