ANTIMICROBIAL ACTIVITY OF TERMINALIA BELLERICA (GAERTN.) ROXB. AGAINST MULTIDRUG-RESISTANT STAPHYLOCOCCUS SAPROPHYTICUS

ANTIMICROBIAL ACTIVITY OF TERMINALIA BELLERICA (GAERTN.) ROXB. AGAINST MULTIDRUG-RESISTANT STAPHYLOCOCCUS SAPROPHYTICUS

Tamalika Chakraborty, Sumana Chatterjee *, Lopamudra Datta and Abhijit Sengupta

Department of Pharmacy, Guru Nanak Institute of Pharmaceutical Science and Technology, 147/F, Nilgunj Road, Panihati, Kolkata, West Bengal, India.

ABSTRACT: The present study investigates the isolation and identification of Multi-Drug Resistant bacteria from Hospital Effluent and the determination of Antimicrobial activity of Terminalia bellerica (Roxb.) against the isolated Multi Drug-Resistant Bacteria. Hospital effluent was collected and screened for the presence of Drug resistance phenotype against five different antibiotics, and the isolated bacterium was identified by 16s rRNA sequencing. Terminalia bellerica Roxb., Baheda has established antimicrobial activity against Gram-negative and Gram-positive bacteria hence the above plant was selected for the present investigation. Ethanolic extract of Terminalia bellerica outer coat, fruit, and the seed was tested for antimicrobial activity against the isolated Multi Drug-Resistant Bacteria, and the MIC₉₀ value was determined. Ethanolic extract of T. bellerica outer coat having the highest zone of inhibition was further fractionated, and four individual fractions, F1 (water), F2 (50% ethanol and water), F3 (ethanol), and F4 (acetone) were tested for antimicrobial activity against isolated Multi Drug-Resistant Bacteria. Staphylococcus saprophyticus, which was isolated from Hospital effluent and identified by 16 s-rRNA sequencing, was found to be resistant to five antibiotics with the highest resistance against Cefixime MIC₉₀ 24 ± 0.00 (µg/ml). Crude extract of Terminalia bellerica outer coat obtained by maceration gave the highest zone of inhibition of 3.4 ± 0.17(cm). Fraction F4 gave highest zone of inhibition 3.2 ± 0.17 (cm) with MIC₉₀12 ± 0.00 (µg/ml). The extractive value and yield of fraction F4 are 1500mg and 0.030%. Phytochemical test of F4 was found to contain tannin and alkaloid.

Keywords: Antimicrobial, Screening, Multi-Drug Resistance, Phytochemicals, Purification

INTRODUCTION: Antibiotics are boon to society as these drugs have potentially reduced the incidence of infectious diseases and death due to them. Antibiotics can be effective when prescribed correctly and used rationally, but unfortunately, the present scenario is different ¹. There is the emergence of Antibiotic-resistant bacteria, which is a present threat to the environment and society.

Antibiotic resistance stems from the abuse and irrational use of antibiotics; moreover, antibiotic resistance among bacteria can spread over several Genera making sensitive bacteria resistant ².

Some bacteria are resistant to a single antibiotic, whereas others are resistant to multiple different antibiotics and are often referred to as Multi Drug-Resistant Bacteria (MDR). There are several groups of bacteria that have been proved to be a potential MDR, such as MRSA (Methicillin-Resistant Staphylococcus aueus), Escherichia coli, Haemophilus sp and many other β- Lactamase producer. Pseudomonas aeruginosa is known to have multidrug efflux pump, which can forcefully expel nonspecific antibiotics out of the cell, whereas Escherichia coli has accumulated many antibiotic-resistant gene in a cassette located in an R(resistant) plasmid ^{1, 3-4}. The present scenario demands a new antimicrobial agent to combat multidrug-resistant bacteria. There are many newly synthesized antimicrobial agents of chemical origin but with a short life expectancy; hence, herbal products from Indian Medicinal Plants can be an alternative remedy for the present problem. A new lead molecule of herbal origin needs to be identified which will be a potential antimicrobial agent or a good resistance modifier ^{5, 6.}

Terminalia bellerica belongs to the family “Combretaceae” and is commonly known as belleric Myrobalan is a fast-growing deciduous tree with a rounded crown. It can even grow up to 50 meters ⁷. The bark is bluish or ash-grey, whereas the inner bark is yellow. Fruits are sub-globular to broadly ellipsoid, light yellow, and have brown tomentosa. Leaves are found to belong, oval, long, and usually clusters towards the end of the branch. Flowers are white and yellow with an offensive odour. It is also called as Baheda in India and has been long used in Indian Herbal Medicine. It is anti-helminthic, digestive, laxative and astringent useful in ailments like cough, diarrhoea, dyspepsia, asthma, bronchitis etc. ^{8, 9} Many Phyto-constituents have been isolated from the fruit such as Anthraquinones, Chebulagic acid, Gallic acid etc.; hence, this research is an attempt to study the antimicrobial activity of T. bellerica fruit against Multi Drug Resistant Staphylococcus saprophyticus which is responsible for community-acquired urinary tract infection.

METHODS:

Source of the Pathogen: Effluent from a Dental hospital (Guru Nanak Institute of Dental Science and Research, Sodepur) was collected for the isolation of MDR.  Hospital effluent was collected because many antibiotics were applied to the patient during surgery and other treatments, which were ultimately discharged into the wastewater, and hence, the probability of getting a drug-resistant pathogen was more.

Isolation of Microbial Pathogen: The sample was subjected to serial dilution in 0.9% Saline and plated in Nutrient Agar using Pour Plate Technique for isolation of individual colony. The colonies obtained after incubation at 37 °C for 24 h were further screened for antibiotic resistance ¹.

Screening of Colonies for Antibiotic Resistance: Two well-distinguished colonies having opaque white and golden yellow colour respectively were selected for further investigation and hence, pure cultures were prepared in slant as well as broth. The focus was given to the Golden yellow colony as Staphylococcus species. Produces Golden yellow colony and, moreover, there is an increased emergence of MRSA (Methicillin-Resistant Staphylococcus aureus) ^{6, 10}.

Antibiotics Susceptibility Profiling of the Isolated Colonies: The above-selected colonies were screened for antibiotic resistance against five following antibiotics, namely Azithromycin, Clarithromycin, Amoxicillin, Cefixime, and Tetracycline. Considering the MIC₉₀ according to Clinical and Laboratory Standards Institute (CLSI) guidelines 2016, following concentrations of 4 µg/ml, 8 µg/ml, 12 µg/ml, 16 µg/ml, 24 µg/ml and 30 µg/ml were prepared. All the tests were performed in Muller Hinton (HI-Media) Agar Plates ^{11, 12-13}.

Biochemical Characterization of Selected Colony Following Bergey’s Manual: The selected colony which was found to be resistant was subjected to morphological identification using Gram’s staining method followed by its bio-chemical characterization through various Bio-chemical tests such as Catalase, Oxidase, Glucose Fermentation, and Lactose fermentation, Mannitol Fermentation, Methyl red, Voges Proskauer and Indole test. Staphylococcus sp. was finally confirmed by growing in Blood agar and determining of nature of its hemolysis. The pathogenicity of Staphylococcus species was determined by performing Coagulase Test. All the tests were performed according to Standard Bergey’s Manual of Bacteriology to characterize the microorganism accurately ^{14, 15}.

Identification of Staphylococcus sp by 16s rRNA Sequencing: DNA was isolated from the isolated culture. The DNA quality obtained was evaluated on Agarose Gel of 1.0% and found to have one single band. Fragment of the 16S rDNA gene was further amplified by 27F using 1492R primers. Further analysis resulted in a single discrete band of 1500bp when resolved in Agarose Gel. The PCR (Polymerase Chain Reaction) amplicon was purified to remove contaminants. Forward and reverse DNA sequencing reaction of PCR amplicon was carried out with forwarding primer and reverse primers using BDT v3.1 Cycle sequencing kit with ABI 3730xl Genetic Analyzer. A consensus sequence of the 16S rDNA gene was generated from forward and reverse sequence data using aligner software. The 16S rDNA gene sequence was used to carry out BLAST with the database of NCBI GenBank database. A maximum identity score was obtained and the first ten sequences are further selected and subjected to alignment using Clustal W, a multiple alignment software. A distance matrix was generated, and the phylogenetic tree was constructed using MEGA 7.

Preparation of Plant Extract: Fresh dry fruit of Terminalia bellerica was collected from the areas of North 24 Parganas. The fruit was authenticated from the Central National Herbarium-Botanical Survey of India, Kolkata. The fruit was washed 3-4 times and further dried under shade. The fruit was further crushed and separated into Exocarp (Outer Coat), Endocarp (Fruit) and seed. Each part of the fruit is converted to a fine powder and subjected to extraction.

Extraction Procedure: All the parts of Baheda fruit were extracted by Maceration, whereas the Outer Coat was extracted both by Maceration and Ultrasonic Assisted Extraction (UAE) ¹⁶.

Maceration: 10g of finely grounded powder was taken and dissolved in 100ml of 95% Ethanol. The samples were subjected to shaking at 120 rpm at 25 °C for 7 days until the extraction was complete. The extract was filtered and evaporated in Rotary Vacuum Evaporator at 45 °C until dry. 1 g of ethanol extract was mixed in 5ml of ethanol to give a concentration of 0.2mg/ml

Ultrasonic Assisted Extraction: 5g of Baheda Outer coat was taken and suspended in 95% Ethanol in the ratio of (1: 20 w/v). The content was mixed thoroughly, and the Ultrasonic bath was set at 25 °C for 15 min. Ultra-sonication was done for 30 min. After the completion of extraction, the extract was centrifuged at 3000 rpm for 15 min. The supernatant was collected, filtered and evaporated in Rotary Vacuum Evaporator at 45 °C until dry. 1 g of ethanol extract was mixed in 5ml of ethanol to give a concentration of 0.2mg/ml ¹⁷.

Determination of Antimicrobial Activity of Terminalia bellerica against MDR Staphy-lococcus saprophyticus: Crude extract of Outer coat, fruit, and seed extracted by Maceration and Ultrasonic Assisted extraction was charged against isolated Staphylococcus saprophyticus inoculum volume (0.1 ml) containing 10⁸ cells for determination of antimicrobial activity. The test was performed using the standard Disc Diffusion method using sterile paper discs (5mm diameter). The Test Disc with crude extract, Ethanol as Control, and Cefixime as Standard were placed in a lawn of S. saprophyticus swabbed in Muller Hinton Agar Plates. Bacterial cultures were further incubated at 37 °C for 24 h. Zone of Inhibitions was measured and all the tests were performed in triplicates and the average zone of inhibition was noted ¹⁸.

Phytochemical Study of Terminalia bellerica: The extract with the highest zone of inhibition was selected and phytochemical constituents were identified by various phytochemical tests. The yield and extractive values were calculated according to Indian Pharmacopoeia 2014 ^{19, 20-21, 32}.

Fractionation of Terminalia bellerica Outer Coat: 5g of Ethanolic extract of Baheda Outer Coat was absorbed with 10g of silica, the adsorbed material was washed with 50 ml of Acetone. The Acetone soluble portion was collected, and the insoluble portion was washed with 50 ml 100% Ethanol. Ethanol soluble portion was collected, and insoluble portion was washed with 50% Ethanol/ water followed by 100% demineralized water. The following fractions were dried and the extractive value, yield of the fractions was further determined ²².

Determination of Antimicrobial Activity of Terminalia bellerica Fractions against MDR Staphylococcus saprophyticus: The test was performed using the standard Disc Diffusion method using sterile paper discs (5mm diameter). Test Discs with crude extract of all the four fractions, Ethanol as Control and Cefixime as Standard, were placed in a lawn of S. saprophyticus swabbed in Muller Hinton Agar Plates and the cultures were further incubated at 37 °C for 24 h. Zone of Inhibition was measured and all the tests were performed in triplicates and the average zone of inhibition was noted ²³.

Determination of MIC₉₀ (Minimum Inhibitory Concentration) of Terminalia bellerica Fractions against MDR Staphylococcus saprophyticus: Each fraction was dissolved in 10% DMSO (Dimethyl Sulfoxide) for the determination of Minimum Inhibitory Concentration of the Extract against the isolated MDR. Concentrations of 4 µg/ml, 8 µg/ml, 12 µg/ml, 16 µg/ml, 24 µg/ml, 30 µg/ml were prepared for all the fractions, and the minimum inhibitory concentration was determined against the MDR Saprophyticus ^{24, 25-26}.

Determination of Phytochemicals Associated with Terminalia bellerica Fractions: Phyto-chemical tests for all the fractions were performed namely Ferric chloride test and Gelatin test for Tannins, Shinoda test, Ferric chloride test and Zn/HCL reduction test for Flavonoids, Mayer’s test, and Wagner’s test for alkaloid, Solubility test and filter paper test for Fats, Ninhydrin test and Biuret test for proteins and Salkowski test for steroid ^{19, 20-27, 28}.

RESULTS:

Characterization of Isolated Microorganism from Hospital Effluent:

FIG. 1: GRAM POSITIVE COCCI IN BUNCHES AS OBSERVED IN 100X OBJECTIVE

TABLE 1: BIOCHEMICAL TESTS DONE ACCORDING TO STANDARD BERGEY’S MANUAL

Determination of Phenotypic Antibiotic Resistance against Staphylococcus saprophyticus:

TABLE 2: REFERENCE RANGE OF ANTIBIOTICS SUSCEPTIBILITY OF STAPHYLOCOCCUS SAPROPHYTICUS ACCORDING TO CLSI GUIDELINES 2016

FIG. 2: COMPARATIVE STUDY OF ZONE OF INHIBITION IN (mm) OF 5 ANTIBIOTICS AGAINST ISOLATED STAPHYLOCOCCUS SAPROPHYTICUS

TABLE 3: AVERAGE ZONE OF INHIBITION (mm) OBTAINED FROM CHARGING 5 ANTIBIOTICS AGAINST STAPHYLOCOCCUS SAPROPHYTICUS. AVERAGE ZONE OF INHIBITION OBTAINED BY PERFORMING THE EXPERIMENTS IN TRIPLICATE

TABLE 4: OVERALL RESISTANCE PATTERN OF ISOLATED MDR STAPHYLOCOCCUS SAPROPHYTICUS ACCORDING TO CLSI 2016

Identification of the Isolated Multidrug-Resistant Bacteria: The above specimen showed high similarity with Staphylococcus saprophyticus based on nucleotide homology and phylogenetic analysis.

gDNA and 16S Amplicon QC Data:

Sanger Sequence Chromatogram File Data: >Forward Seq data GGCCAAGATGAATGCTAGTG TTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAATCTTGACATCCTTTGAAAACTCTAGAGATAGAGCTTTCCCCTTCGGGGGACAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTAAGCTTAGTTGCCATCATTAAGTTGGGCACTCTAGGTTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCTACAATGGACAATACAAAGGGCAGCTAAACCGCGAGGTCATGCAAATCCCATAAAGTTGTTCTCAGTTCGGATTGTAGTCTGCAACTCGACTACATGAAGCTGGAATCGCTAGTAATCGTAGATCAGCATGCTACGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGCCGGTGGAGTAACCATTTATGGAGCTAGCCGTCGAAGGTGGGACAAATGATTGGGGTGAAGTCGTAACAAGGTAACCGTAAGC.

>Reverse Seq Data GGGGGGGTCTCCAGGCGGAATGCTTAATGCGTTAGCTGCAGCACTAAGGG GCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGATCCCCACGCTTTCGCACATCAGCGTCAGTTACAGACCAGAAAGTCGCCTTCGCCACTGGTGTTCCTCCATATCTCTGCGCATTTCACCGCTACACATGGAATTCCACTTTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCAATGACCCTCCACGGTTGAGCCGTGGGCTTTCACATCAGACTTAAGAAACCGCCTACGCGCGCTTTACGCCCAATAATTCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGATTAGGTACCGTCAAGATGTGCACAGTTACTTACACATTTGTTCTTCCCTAATAACAGAGTTTTACGAGCCGAAACCCTTCATCACTCACGCGGCGTTGCTCCGTCAGGCTTTCGCCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGTATCGTCGCCTTGGTAAGCCGTTACCTTACCAACTAGCTAATACGGCGCGGGTCCATCTATAAGTGATAGCAAAACCATCTTTCACTTTAGAACCATGCGGTTCTAAATGTTATCCGGTATTAGCTCCGGTTTCCCGAAGTTATCCCAGTCTTATAGGTAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACTTCAAAGGAGCAAGCTCCTTATCTGTTCGCTCGACTTGCATGTATTAGGCACGCCGCCAGCGTTCATCCTGAGCCAGGAATCAAACTCTACGGTTACCTTGTTACGAGTTACTGGGTCAGGATTAAAGAAAACGCTGAAAAGT.

>Reverse complement ACTTTTCAGCGTTTTCTTTAATCCTGACCCAGTAACTCGTAACAAGGTA ACCGTAGAGTTTGATTCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAACAGATAAGGAGCTTGCTCCTTTGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTACCTATAAGACTGGGATAACTTCGGGAAACCGGAGCTAATACCGGATAACATTTAGAACCGCATGGTTCTAAAGTGAAAGATGGTTTTGCTATCACTTATAGATGGACCCGCGCCGTATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGACGATACGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAGTGATGAAGGGTTTCGGCTCGTAAAACTCTGTTATTAGGGAAGAACAAATGTGTAAGTAACTGTGCACATCTTGACGGTACCTAATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGTAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAAACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGCAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACGCTGATGTGCGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGAGACCCCCCC.

>S_AUREUS consensus seq CCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGT CGAGCGAACAGATAAGGAGCTTGCTCCTTTGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTACCTATAAGACTGGGATAACTTCGGGAAACCGGAGCTAATACCGGATAACATTTAGAACCGCATGGTTCTAAAGTGAAAGATGGTTTTGCTATCACTTATAGATGGACCCGCGCCGTATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGACGATACGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAGTGATGAAGGGTTTCGGCTCGTAAAACTCTGTTATTAGGGAAGAACAAATGTGTAAGTAACTGTGCACATCTTGACGGTACCTAATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGTAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAAACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGCAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACGCTGATGTGCGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAATCTTGACATCCTTTGAAAACTCTAGAGATAGAGCTTTCCCCTTCGGGGGACAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTAAGCTTAGTTGCCATCATTAAGTTGGGCACTCTAGGTTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCTACAATGGACAATACAAAGGGCAGCTAAACCGCGAGGTCATGCAAATCCCATAAAGTTGTTCTCAGTTCGGATTGTAGTCTGCAACTCGACTACATGAAGCTGGAATCGCTAGTAATCGTAGATCAGCATGCTACGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGCCGGTGGAGTAACCATTTATGGAGCTAGCCGTCGAAGGTGGGACAAATGATTGGGGTGAAGTCGTAACAAGGTAACCGTA

DATA: (Alignment view using a combination of NCBI GenBank):

Distribution of 100 Blast Hits on the Query Sequence:

Sequences Producing Significant Alignments:

TABLE 5: IDENTIFICATION OF STAPHYLOCOCCUS SAPROPHYTICUS

Phylogenetic Tree:

FIG. 3: MOLECULAR PHYLOGENETIC ANALYSIS WAS BASED ON MAXIMUM LIKELIHOOD METHOD AND THE EVOLUTIONARY HISTORY WAS DETERMINED BY MAXIMUM LIKELIHOOD METHOD FOLLOWING KIMURA 2 PARAMETER MODEL ²⁹

1000 replicates were considered for drawing inference of bootstrap consensus tree and the following data is taken to determine the evolutionary history of the taxa analyzed ³⁰. Branches associated with the partitions reproduce in less than 50% replicates of bootstrap are collapsed. Next to the initial tree(s) comprise of the percentage of replicate trees which are associated with the taxa clustered together in the bootstrap test. The pairwise distance was analyzed and estimated using the Maximum Composite Likelihood (MCL) approach followed by a selection of topology with superior log likelihood value. The above analysis includes 11 nucleotide sequences. The following codon positions were included as 1^st + 2^nd + 3^rd + Non-coding, followed by elimination of positions containing gaps and missing data. The final data set contained 1434 positions, and the evolutionary analysis was performed in MEGA 7 ³¹.

Distance Matrix:

TABLE 6: ESTIMATES OF EVOLUTIONARY DIVERGENCE BETWEEN SEQUENCES

“The number of base substitutions per site from between sequences is shown. Standard error estimate(s) are shown above the diagonal. Analyses were conducted using the Kimura 2-parameter model ²⁹. The analysis involved 11 nucleotide sequences. Codon positions included were 1^st + 2^nd + 3^rd + Noncoding. All positions containing gaps and missing data were eliminated.

There were a total of 1434 positions in the final dataset. Evolutionary analyses were conducted in MEGA7” ³¹.

Antimicrobial Activity of Terminalia bellerica Roxb. against Multi-Drug Resistant Staphylococcus saprophyticus:

FIG. 4: ANTIMICROBIAL ACTIVITY OF TERMINALIA BELLERICA CRUDE EXTRACT AGAINST MDR STAPHYLOCOCCUS SAPROPHYTICUS

FIG. 5: ANTIMICROBIAL ACTIVITY OF 4 DIFFERENT FRACTIONS OF TERMINALIA BELLERICA EXOCARP AGAINST MDR STAPHYLOCOCCUS SAPROPHYTICUS

TABLE 5: PHYTOCHEMICAL CONSTITUENTS OF 4 FRACTIONS OF TERMINALIA BELLERICA EXOCARP

TABLE 6: EXTRACTIVE VALUE AND YIELD OF TERMINALIA BELLERICA FRACTIONS

FIG. 6: COMPARATIVE ZONE OF INHIBITION STUDY OF ALL 4 FRACTIONS OF TERMINALIA BELLERICA EXOCARP TAKING CEFIXIME AS STANDARD

FIG. 7: COMPARISON OF MIC OF T. BELLERICA FRACTION (EXOCARP) WITH RESPECT TO STANDARD CEFIXIME

DISCUSSION: Microscopic examination of the isolated bacteria from effluent was found to be Gram-positive Coccus, looked like a bunch of grapes under 100 x objectives, which was characteristics of Staphylococcus species Fig. 1. The following microorganism was further characterized by the Biochemical tests according to the Bergey’s Manual. Microorganism was found to be positive for the following tests such as Catalase, Oxidase, Lactose Fermentation, Mannitol Fermentation, Methyl Red, Voges Proskauer and Indole test Table 1. The above results proved that the microorganism was Staphylococcus sp. It was further confirmed by growing the bacteria in blood agar showing no hemolysis (Gamma reaction). The bacteria were found to be pathogenic as it was Coagulase-negative as well; the organism was further confirmed and identified by 16 s rRNA sequencing; hence, it was concluded from the above evidence that the isolated bacterium was none other than a pathogenic Staphylococcus saprophyticus Table 7. Antibiotics were charged against the isolated Staphylococcus saprophyticus, and the bacteria were found to be resistant to 5 different antibiotics such as Azithromycin, Clarithromycin, Amoxicillin, Cefixime and Tetracycline Fig. 2, Table 4. The Minimum Inhibitory Concentration (MIC) of the antibiotics were compared with the standard MIC according to CLSI Guidelines 2016 Table 2. The bacteria were proved to be Multi-Drug Resistant with the highest resistance against Cefixime MIC₉₀24+0.00 µg/ml Table 3, Table 4, Fig. 3.

Terminalia bellerica fruit was selected for the screening of antibacterial activity against Multi-Drug Resistant Staphylococcus saprophyticus and was found to have potent antibacterial activity against isolated MDR S.saprophyticus. Fruit of T. bellerica showed good antimicrobial activity against the MDR and the highest activity was shown by the exocarp extracted by maceration with zone inhibition of 3.4 ± 0.17cm Fig. 4. The Exocarp extract with the highest antibacterial property was further fractionated to separate the phytochemical constituents responsible for the activity. The acetone fraction of the exocarp gave the highest antimicrobial activity against the isolated MDR with a zone of inhibition of 3.2 ± 0.17 cm Fig. 5. All, the fractions were tested for phytochemical constituent Table 5. Phytochemical constituents present in F4 fraction were Tannin Alkaloid and Steroid. Minimum Inhibitory Concentration of all the fractions against MDR S saprophyticus was determined and compared with standard Cefixime (with the highest MIC). Comparison of MIC of 4 purified fractions of T. bellerica exocarp with respect to Cefixime showed acetone fraction has the highest antimicrobial activity with the lowest MIC-12 ± 0 µg/ml and Cefixime with MIC 24 ± 0µg/ml Fig. 5, Fig. 6. The extractive value and Yield of all the fractions were determined in Table 6. The P-value for the MIC of T. bellerica fractions obtained by ONE WAY ANOVA was P<0.0001, which was statistically significant.

CONCLUSION: Staphylococcus saprophyticus is associated with uncomplicated Urinary Tract Infection (UTI) in humans. It is one of the leading causes of cystitis in young women. The organisms are also associated with some severe complications like pyelonephritis; septicemia, nephrolithiasis, and endocarditis.  Antibiotics are the only choice for treating those diseases. Unfortunately, if Multi-Drug Resistant pathogenic Staphylococcus saprophyticus thrives in the environment, then it can cause a severe form of the above diseases with no fruitful treatment and the ultimate consequence will be increased mortality in the population; hence, an alternative remedy is required to combat Multi Drug Resistance. The above study revealed a potential antimicrobial activity of acetone fraction of T. bellerica outercoat against isolated multi-drug resistant Staphylococcus saprophyticus. The acetone fraction comprises Alkaloids and tannins as primary phytochemicals. This work can be further proceeded by identification and characterization of the active phytoconstituents of T. bellerica exocarp, which can have an antibacterial property or can be good resistance modifier for Multi Drug-Resistant Bacteria. Mechanism of resistance of the above bacteria also need be determined for the formulation of potential therapeutic agent against MDR Staphylococcus saprophyticus.

ACKNOWLEDGEMENT: The authors are grateful to the Institution Guru Nanak Institute of Pharmaceutical Science and Technology for all the support and the lab facilities provided to complete the research work.

CONFLICTS OF INTEREST: There is no potential conflict of interest.

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    How to cite this article:

    Chakraborty T, Chatterjee S, Datta L and Sengupta A: Antimicrobial activity of Terminalia bellerica (Gaertn.) Roxb. against multi drug resistant Staphylococcus saprophyticus. Int J Pharm Sci & Res 2021; 12(10): 5353-63. doi: 10.13040/IJPSR.0975-8232.12(10).5353-63.

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