Effect of Ginkgo biloba and Anacyclus pyrethrum against Streptococcus species isolated from Dental caries and Periodontitis

Usha Subbiah¹*, Athira Ajith¹, Harini Venkata Subbiah¹, Sonaa Elango²

¹Human Genetics Research Centre, Sree Balaji Dental College and Hospital,

Bharath Institute of Higher Education and Research, Chennai, Tamil Nadu, India.

²Department of Life Science, School of Natural Sciences, University of Suwon,

Wau-ri, Bongdam -eup, Hwaseong-si, Gyeonggi - do, Republic of Korea.

*Corresponding Author E-mail: ushas@sbdch.bharathuniv.ac.in, ushat75@yahoo.com, athiramtdy7@gmail.com, harini.venkatt@gmail.com, sonaa.elango@gmail.com

ABSTRACT:

Streptococcusis the main etiological agents of dental caries and periodontitis. This study aims to evaluate the antimicrobial activity of Anacyclus pyrethrum and Ginkgo biloba extracts on the isolated Streptococcus speciesfrom dental caries and periodontitis samples. Antimicrobial susceptibility assays such as the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extract against Streptococcus species were performed. S. mutans resistance was also determinedby plasmid curing. The extract of A. pyrethrum and G. biloba exhibited 82.55% and 81.73% of inhibition against Streptococcus mutans at MIC 8 mg/ml and 9mg/ml respectively. Other Streptococcus species are resistant to A. pyrethrum (>77%) and G. biloba (>74%). MBC of A. pyrethrum extract was 20mg/ml for S. mutans and 22mg/ml for other Streptococcus species. Meanwhile, MBC was 24mg/ml and 26mg/ml for G. bilobaagainst S. mutansand other Streptococcus species respectively. The plasmid curing by faint plasmid band analysis indicated that these herbal extracts partially reversed the resistance against S. mutans. Our observations indicated that the A. pyrethrum and G.biloba extracts combat resistance against oral streptococcus. Further study needs to be conducted to check the potential active ingredients of these herbal extracts and to be formulated in oral care applications.

KEYWORDS: Streptococcus, resistance, Plasmid curing, Anacyclus pyrethrum, Ginkgo biloba.

INTRODUCTION:

Oral health is vital to general fitness and well-being at all stages of life. Dental caries and periodontal illness are amongst the most prevalent oral diseases in mankind. Dental caries is a ubiquitous process involved in the chemical dissolution of the tooth surface¹. Periodontitis,a chronic disease caused by bacterial plaque that introduces a host immuno-inflammatory response on the hard and soft tissue structures around the teeth. The oral microbiome implicates the development and progression of both caries and periodontitis².

Streptococcus mutans is the main cause of dental decay thereby detachinggums from the teeth as a result of an inflammatory response to dental plaque. The Streptococcusspecies that are primarily involved in initial dental plaque formation are S. mutans, S. sanguinis, S. mitis, S. gordonii, and S. oralisare also associated with the risk of systemic diseases and invasive infections³.

For the prevention and control of dental caries and periodontal diseases, many prophylactic agents have been used for years. For instance antibiotics, mouthwashes, toothpastes, gels, varnishes, and plant derived compounds arealso used for the treatment⁴. Anacyclus pyrethrum is a traditional herbal plant commonly called Akarkara, having the benefits of toothache alleviation, prevention from tooth decay and treatment of periodontal diseases has been widely used in the Indian ayurvedic medicinal system. Studies showed that the root of A. pyrethrum is a strong sialagogue, its root powder prevents tooth decay progression and treating gingival diseases in Iranian medicine⁵. Traditionallythese plants have antibacterial, anti-inflammatory effects, tonic to the nervous system and positive effects on regulating the immune system⁶. Ginkgo biloba has been extensively used in traditional Chinese medicine. The crude organic extracts of leaves of it collected at high altitude in Kumaun Himalaya, India, has high antibacterial potential against a wide range of pathogenic bacterial strains⁷. Ginkgolic acids are considered to be the major compounds having several pharmacological effects such as antitumor, antidepressant, and antimicrobial properties⁸. Chinese experimental studies on the effect of Ginkgo biloba extract on oral cariogenic bacteria found that it can inhibit Streptococcus mutans⁹.

Antibiotic resistance is highly influenced by the genetic foundation of resistance. Plasmids carry multiple antibiotic resistance genes and contribute to spreading multidrug resistance by allowing pathogenic bacteria to pick up numerous resistance genes in a single transfer event. The plasmid transmission of antibiotic resistance can be eliminatedfrom their hostsif the resistance genes are harbored in plasmid but not in chromosome. The purpose of this study was to investigate the antimicrobial resistance and plasmid curing effect of Anacyclus pyrethrum and Ginkgo biloba extracts against S. mutansandother Streptococcus sp. isolated from dental caries and periodontitis.

MATERIALS AND METHODS:

Study Design:

Chronic Periodontitis (n= 5) and dental caries (n=5) selected for our study were outpatients in the Department of Periodontics and Department of Conservative Dentistry and Endodontics, Sree Balaji Dental College and Hospital, Chennai, India. Written informed consent was obtained from all the cases. The study was approved by the Ethical Committee Institutional Review Board (SBDCH/IEC/09/2016/20) and (SBDCH/IEC/12/2019) with the following Inclusion and exclusion criteria.

Inclusion Criteria:

Adult participants aged between 18-30years. Patients with 12 scoreable teeth who have chronic periodontitis. Patients with dental caries of DMFT index ≥ 5 in the age group of 25-60 were included.

Exclusion Criteria:

Alcoholism, smokers or former smokers who quit within the last 5 years, pan/gutka/tobacco chewers, phenytoin, calcium channel blockers, cyclosporin users, aggressive periodontitis within the last 6 months to a year, antibiotic therapy or immunosuppressantmedication within the last 6 months, scaling and polishing within the last 30 days, pregnancy or lactation.

Whole saliva and subgingival plaque samples were collected. The deepest pocket of each quadrant was preferred for subgingival plaque sampling. The samples were pooled with phosphate buffer saline and frozen at -20°C before analysis.

Preparation of Extracts of Anacyclus pyrthrum and Ginkgo biloba:

Anacyclus pyrethrum was collected in the region of Nagercoil, Tamil Nadu, India and was authenticated and certified (NISMB3322018) by the Department of Medicinal Botany, National Institute of Siddha, Chennai, India. The roots were removed from freshly collected plants of A.pyrethrum and then cleaned, shade dried, powdered and steeped in ethanol for 48h. To make it in powder form, the herbal extract was filtered through a 90mm filter paper and dried at 50°C. The yield of ethanolic extract for A.pyrethrum was 28.5%²⁰. Briefly, Ginkgo biloba leaves were collected from the Nainital district of Uttarakhand. The leaves were washed and air-dried for several weeks and were ground to a fine powder and kept at room temperature in air tight bottles. For aqueous extraction, 10g of leaf powder was mixed in 100ml of distilled water, boiled for 6h to concentrate until the final volume became 1/4th of the initial. The residue was filtered through Whatman filter paper no. 42 after 2hrs. Finally, 10g of material (pulp) were extracted in 25ml of distilled water and stored at 4° C.

Isolation of Streptococcal Species:

Subgingival plaque sample in PBS was vortexed for a minute to disperse the plaque in suspension.10μl of the plaque suspension sample was streaked on MS-agar (Mutans Sanguis agar) plates and incubated for 48hours at 37°C in a candle-jar that provide ~5% CO₂. Colonies grown on MS-agar medium were again streaked on the blood agar plates and incubated anaerobically for 48h to isolate the pure Streptococcus sp. The pure subculture from each sample plate was transferred to brain–heart infusion (BHI) broth (Hi Media, India) and incubated at 37°C for 18h.

Standard morphological and gram staining was performed in our laboratory for preliminary phenotypic identification of the isolates, according to the information in Bergey’s Manual of Determinative Bacteriology¹⁰.

Molecular Identification:

16S rDNA was obtained by boiling lysis method¹¹, with slight modifications. Brieflya single colony was dispersed in 100μl of nuclease free water. The suspension was boiled at 100°C for 15min and kept immediately at -20°C for 5 minutes followed by centrifugation at 13,000rpm for 10 minutes. The lysate (4μl) was used as template for the amplification of 16S rDNA in 20μl of reaction mixture including 1μl of 10 picomoles of each primer -16S forward primer (27F) (5’‑AGAGTTTGATCMTGGCTCAG‑3’) 16S reverse primer (1492R) (5’‑GGTTACCTTGTTACGACTT‑3’). The PCR reaction conditions for 34 cycles were as follows: Initial denaturation 94°C for 2 min, denaturation 94°C for 1min, annealing 58°Cfor 2 min, extension 72°C for 1min, and final extension 72°C for 15min after standardization using various annealing temperature 52°C to 60°C.

Determination of Minimum Inhibitory Concentration (MIC):

The MIC profile of Anacyclus pyrethrum, Ginkgo biloba extracts and Ampicillin was determined by the macro dilutionmethod. From the stock solution of 100mg/mlofA. Pyrethrumand G. biloba and 10mg/ml of Ampicillin was added in different concentrations to 5ml of BHI broth. A single colony Streptococcus from the cultured agar plates were incubated at 37°C for 24 h. The experiment was performed in triplicates to reproduce the results. The percentage of bacterial inhibition by each extract was as follows:

Optical density in control- Optical density in test set

Percentage = --------------------------------------------- ×100

Inhibition Optical density in control

Minimum Bactericidal Concentration (MBC):

The lowest concentration of the extract that completely suppresses bacterial growth was determined by MBC. 100μl of the culture that was utilized for the MIC was considered for MBC. The agar plate was incubated at 37°C for 24h. To determine the MBC, the absence of bacterial growth on the agar plate was evaluated.

Plasmid isolation:

The S. mutansculture that was used for the determination of MIC was selected for plasmid isolation using Ampicillin, A. pyrethrum and G. biloba. Plasmid isolation was carried out from respective isolates according to the alkali lysismethod of Sambrooket al.,¹²and plasmid curing was confirmed by 0.8% agarose gel electrophoresis.

Determination of antibiotic resistance:

Antibiotic dilutions of 10mg/ml were prepared using Tetracycline, Erythromycin, Ampicillin, Neomycin, Levofloxacin, Amoxicillin, Norflox, Azithromycin, Rifampicin, Oflaxacin, Gentamicin, Cephalosporin, Trimethoprim, Amikacin, Streptomycin, Doxycillin, Cefixime, and Ciprofloxacin. 5μl of each antibiotic was added to each tubecontaining5ml of Muller Hinton Broth and 5μl of overnight cultured isolates and the tubes were incubated at 37°C for 24h. The growth of each isolate on the different antibiotic dilutions was monitored.

RESULT AND DISCUSSION:

Morphological Identification:

The bacteria grown on selective Mutans Sanguis Agarwas Streptococcus group and inhibited other bacterial species growth. Colony morphology resembling S. mutans in the MS agar was identified morphologically as described by Facklam (1977) and Coykendall (1989)^13,14. Streptococcus mutans has a raised, convex, undulate, opaque, granular frosted glass appearance that distinguishes it from other species. The other streptococcal species was identified by glossy, grayish white, small colonies like a twisted bunch of round berries as shown in Figure 1.Streptococci are gram-positive bacteria that are spherical or ovoid in shape and occur in pairs or chains (Figure 2).

(a) (b)

Figure 1: a) MSA agar plate with S. mutans from dental caries saliva sample. b) MSA agar plate with Streptococci sp. isolated from periodontitis subgingival plaque sample.

(a) (b)

Figure 2: Gram staining of oral streptococci. Gram positive and occurring in pairs or chains (a) S. mutans (b) Streptococci sp.

Molecular Identification:

Among the bacterial isolate, S. mutans (3 nos.) and other Streptococcus sp (7nos.) (S. anginous (2), S. pyogenes (3), S. salivarius (1), S. sobrinus (1)) were identified based on 16S rDNA sequencing. The acquired nucleotide sequences were compared to legitimate reference sequences using the BLAST tool, which revealed >99% sequence identity.

Minimum Inhibitory (MIC and MBC) Analysis:

The antibacterial activity of A. pyrethrum and G. biloba extract against S. mutans (3 nos.) and other Streptococcus sp (7 nos.), Ampicillin (standard) was examined in the present study and their potency was quantitatively assessed by MIC and MBC values. The MIC observed for Ampicillin was 10𝜇g/ml and the percentage of inhibition was 96.32%. The MIC against S. mutans for A. pyrethrum and G. bilobaextract was 8 and 9mg/ml (Table 1) and the average percentage of inhibition was 82.55% and 81.73% (Table 2) respectively.

Table 1: Antibacterial activity of Anacyclus pyrethrum extract and Ginkgo biloba on Streptococcus mutans

Herbal Extract

1 mg/ml

mg/ml

4 mg/ml

mg/ml

10 mg/ml

Anacyclus pyrethrum

MIC-

Ginkgo biloba

MIC-

Table 2: Percentage of Streptoccusmutans inhibition at MIC concentration of Anacyclus pyrethrum extract, Ginkgo biloba extract and Ampicilin

Organism	*Anacyclus pyrethrum* extract			*Ginkgo biloba* extract			With ampicilin
Organism	8mg/ml	9mg/ml	10mg/ml	8mg/ml	9mg/ml	10mg/ml	10mg/ml
S. mutans OD	0.321	0.282	0.250	0.342	0.29	0.261	0.06
% of inhibition	80.30	82.69	84.66	79.01	82.20	83.98	96.32
Average % of inhibition	82.55			81.73			-

Table 3: Percentage of inhibition for Streptoccus sp. (7) [S. anginous(2), S. pyogenes(3), S. salivarius, S. sobrinus] at MIC concentration of Anacyclus pyrethrum extract, Ginkgo biloba extract and Ampicilin.

Organism		*Anacyclus pyrethrum* extract			*Ginkgo biloba* extract			With ampicilin
Organism		8mg/ml	9mg/ml	10mg/ml	8mg/ml	9mg/ml	10mg/ml	10mg/ml
*S. anginous*	OD	0.345	0.301	0.253	0.430	0.397	0.360	0.07
*S. anginous*	% of inhibition	72.4	81.53	84.48	73.61	75.64	77.91	95.70
*S. anginous*	OD	0.378	0.299	0.245	0.465	0.401	0.392	0.07
*S. anginous*	% of inhibition	76.80	81.656	84.96	71.47	75.4	75.95	95.70
*S. pyogenes*	OD	0.403	0.397	0.311	0.456	0.389	0.309	0.06
*S. pyogenes*	% of inhibition	75.28	75.64	80.92	72.02	76.13	81.04	96.32
*S. pyogenes*	OD	0.356	0.293	0.256	0.415	0.360	0.317	0.06
*S. pyogenes*	% of inhibition	78.16	82.02	84.29	74.54	81.6	80.67	96.32
*S. pyogenes*	OD	0.326	0.285	0.239	0.423	0.391	0.370	0.08
*S. pyogenes*	% of inhibition	80	82.51	85.34	74.05	76.01	77.3	95.09
*S. sobrinus*	OD	0.321	0.292	0.246	0.450	0.354	0.299	0.07
*S. sobrinus*	% of inhibition	80.30	82.08	84.90	72.39	78.28	81.65	95.70
*S. salivarius*	OD	0.355	0.263	0.211	0.411	0.312	0.309	0.06
*S. salivarius*	% of inhibition	78.22	83.86	87.05	74.78	80.85	81.04	96.32

Table 4: MBC of Anacyclus pyrethrum extract and Ginkgo biloba on Streptococcus mutans

Herbal Extract

mg/ml

16 mg/ml

18 mg/ml

mg/ml

24 mg/ml

mg/ml

Anacyclus pyrethrum

MBC

Ginkgo biloba

MBC

Table 5: Antibiotic resistance of Streptococcus species on different antibiotics at 10mg/ml concentration with listed OD values.

Antibiotics	*S. mutans*	*S. anginous*	*S. pyogenes*	*S. salivarius*	*S. sobrinus*
Tetracycline	0.5	0.12	0.48	0.4	0.38
Erythromycin	0.48	0.43	0.5	0.31	1
Ampicillin	0.06	0.06	0.06	0.25	0.06
Neomycin	0.35	0.21	0.46	0.3	0.46
Levofloxacin	0.29	0.14	0.12	0.25	1
Amoxicillin	0.04	0.04	0.09	0.07	0.15
Norflox	0.25	0.16	0.2	0.2	0.32
Azithromycin	0.14	0.2	0.18	0.1	0.12
Rifampicin	0.36	0.5	0.2	0.12	0.25
Oflaxacin	0.12	0.43	0.32	0.65	0.06
Gentamicin	0.42	0.45	0.5	0.38	0.1
Trimethoprim	0.1	0. 25	0.37	0.25	0.5
Amikacin	0.46	0.47	0.38	0.36	0.1
Cefixime	0.32	0.06	0.12	0.12	0.25
Ciprofloxacin	0.02	0.12	0.06	0.08	0.12

Plasmid Elimination:

The plasmid DNA bands size was approximately ˃5 kb for S. mutans,isolated from caries and periodontitis. The other streptococcus sp., the plasmid band size was not clear for S. pyogenes and S. salivarius (data not shown). The plasmid profile for S. mutans is represented in Figure 3.

Figure 3. Plasmid curing profile for S. mutans. Lane 1 to 5: 50kb DNA marker, S. mutans with A. pyrethrum, S. mutans with G.biloba, S. mutans with Ampicillin, S. mutans.

The plasmid curing for S. mutans with the extract of A. pyrethrum showed a faint plasmid band and G. biloba treated S. mutans also showed a similar faint band. But the Ampicillin treated S. mutans showed a less faint band as compared to A. pyrethrum and G. biloba treated S. mutans.

DISCUSSION:

In dentistry, herbal extracts have been widely used as antimicrobials for reducing inflammation. Studies reported that Anacyclus pyrethrum contains anacycline, isobutylamide, inulin and a trace of essential oil and can be used to decrease the plasma glucose and serum cholesterol levels after oral administration for 3–6 weeks¹⁵. The protective antioxidant activity of Ginkgo biloba with theactive constituentsquercetin, isorhamnetin and kaempherol from long-term diabetic complications such as diabetic nephrotoxicity¹⁶. Streptococcus has been widely analysed against several herbals. From our study it is understood that Ginkgo biloba and Anacyclus pyrethrum extracts showed antimicrobial activity against Streptococcus tested. Plasmid curing indicated that these herbal extracts partially reversed the resistance against S. mutans, andit is indicated that the A. pyrethrum and G.biloba extracts combat resistance against oral streptococcus.This agrees with the report that some of the multidrug-resistant isolates had plasmid while others did not, despite that they were all multidrug-resistant¹¹. Our study indicates that some of the multidrug resistant isolates harbored resistance plasmids which probably must have been acquired. The resistance pattern and strain type of a disease differ by geographical region and information about the local resistance pattern aid clinicians in prescribing antibiotics appropriately¹⁷ and also through the exchange of genetic materials between the plasmid of the same species¹⁸. The plasmid size that was observed in our study was ˃5kb for S. mutans was also reported¹⁹ and other Streptococcussp plasmid size was approximately <5kb and >2kb²⁰. The present plasmid curing indicated that these herbal extracts partially reversed the multi-drug resistance and also supportedthe inhibitory effect of A. pyrethrum and G. bilobaagainst S.mutans. To obtain complete curing of the plasmid, the herbal extracts and/or active biomolecules of these extracts need to be analysed even at higher concentrations. These findings offered the possibility of a combination between antibiotics and herbal extracts and have the potential to disseminate multiple drug-resistant oral pathogens. S.mutans showed maximum resistance for tetracycline and erythromycin which was confirmed from the report that the S. mutans are chromosomally encoded with plasmids conferring tetracycline and erythromycin resistance²¹. Meanwhile S. anginous confers rifampicin and streptomycin harboring plasmid²² and hence from our analysis,
S. angionus showed greater resistance against rifampicin. In the case of S. pyogenes, erythromycin exhibited higher resistance as reported²³. The extracts of A. pyrethrum and G. biloba showed a commendable curing effect and antimicrobial activity and could be preferred to prevent tooth decay and inflammations in dental disease progression.

CONCLUSION:

From the study, we observed that the A. pyrethrum showed more susceptibility against S. mutans and other Streptococcus species than the extract of G. biloba. This could be an effective alternative to the antibiotics used for preventing and controlling dental caries, periodontitis, and other plaque-induced dental diseases. These extracts can be considered for the preparation of herbal mouthwashes and other oral health care products. The herbal extracts of A. pyrethrum and G. biloba can be employed as an antibacterial agent since they have plasmid curing effect. Further studies may be required for the characterization of the potential active ingredients of these herbal extracts before therapeutic application for the development of novel agents that can reduce the overall burden of oral diseases.

DECLARATION OF COMPETING INTERESTS:

The authors declare that they have no competing of interests that could have appeared to influence the work reported in this paper.

FUNDING:

There was no funding received from any funding body or agency for this research.

ACKNOWLEDGEMENT:

Authors wish to thank DST-FIST (Ref. No.SR/FST/College-23/2017), Government of India, New Delhi, India, for utilizing the funded research equipment facilities of SreeBalaji Dental College and Hospital, Pallikaranai, Chennai, Tamil Nadu, India. This study was supported by SreeBalaji Dental College and Hospital, Bharath Institute of Higher Education and Research, Chennai, Tamil Nadu, India. We would like to thank Dr. GeetaTewari, Department, Chemistry, D.S.B. Campus, Kumaun University, Nainital, Uttaranchal, India for gifting Ginkgo biloba aqueous extract.

REFERENCES:

1. Fejerskov O, Nyvad B, Kidd E, editors. Dental caries: the disease and its clinical management. 2015;7-10 John Wiley & Sons,ISBN: 978-1-119-06177-9

2. Sanz M, Beighton D, Curtis MA, et al. Role of microbial biofilms in the maintenance of oral health and in the development of dental caries and periodontal diseases. Consensus report of group 1 of the Joint EFP/ORCA workshop on the boundaries between caries and periodontal disease. J Clin Periodontol. 2017;44:S5-11. https://doi.org/10.1111/jcpe.12682

3. Kim SL, Gordon SM, Shrestha NK. Distribution of streptococcal groups causing infective endocarditis: a descriptive study. Diagn Microbiol Infect Dis. 2018;91(3):269-272. https://doi.org/10.1016/j.diagmicrobio.2018.02.015

4. Chen F, Wang D. Novel technologies for the prevention and treatment of dental caries: a patent survey. Expert Opin Ther Pat. 2010;20(5):681-694. https://doi.org/10.1517/13543771003720491

5. Jalayer-Naderi N, Niakan M. The antibacterial activity of methanolic Anacyclus pyrethrum and Pistacia lentiscus L. extract on Escherichia coli. Iran J Microbiol. 2016;8(6):372. https://sid.ir/paper/315439/en

6. Tyagi S, Ashim MM, Narendra KS, Manoj KS, Bhardwaj P, Singh RK. Antidiabetic effect of Anacyclus pyrethrum DC in alloxan induced diabetic rats. Eur J Biol Sci. 2011;3(4):117-120.

7. Sati SC, Joshi S. Antibacterial activities of Ginkgo biloba L. leaf extracts. Sci World J. 2011;11:2237-2242. https://doi.org/10.1100/2011/545421

8. He J, Wang S, Wu T, Cao Y, Xu X, Zhou X. Effects of ginkgoneolic acid on the growth, acidogenicity, adherence, and biofilm of Streptococcus mutans in vitro. Folia Microbiol (Praha). 2013;58(2):147-153. https://doi.org/10.1007/s12223-012-0191-9

9. Cheng Q, Liu J, Chen D. Experimental study of effect of extract ginkgo biloba on peripheraic blood lymphocyte of recurrent oral ulcertion in rats. J Mod Stomatol. 2006;(3):22.

10. Holt, J. G., Krieg, N. R., Sneath, P. H., Staley, J. T., & Williams, S. T. 1994; 786-788, Bergey's Manual of determinate bacteriology, Williams and Wilikins, Baltimore.

11. Magesh H, Kamatchi C, Vaidyanathan R, Sumathi G. Identification of plasmid-mediated quinolone resistance genes qnrA1, qnrB1 and aac (6’)-1b-cr in a multiple drug-resistant isolate of Klebsiella pneumoniae from Chennai. Indian J Med Microbiol. 2011; 29(3): 262.https://doi.org/10.4103/0255-0857.83910

12. Sambrook J, Fritsch EF, Maniatis T.Molecular Cloning: A Laboratory Manual (2nd ed.). 1989; 182-183, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

13. Coykendall AL. Proposal to elevate the subspecies of Streptococcus mutans to species status, based on their molecular composition. International Journal of Systematic and Evolutionary Microbiology. 1977;27(1):26-30. https://doi.org/10.1099/00207713-27-1-26

14. Facklam RR. Physiological differentiation of viridans streptococci. Journal of Clinical Microbiology. 1977;5(2):184-201.DOI: https://doi.org/10.1128/jcm.5.2.184-201.1977

15. Kushwaha MN, Jatav VS, Pandey S. Plant Anacyclus pyrethrum-A review. Res J Pharmacogn Phytochem. 2012;4(3):164-170.

16. Kale MK, Patil MP, Bhusari KP. Evaluation of Ginkgo biloba in Diabetic Nephrotoxicity. Res J Pharmacogn Phytochem. 2011; 3(6): 286-288.

17. Mohapatra DP, Tiwari P, Debata NK, Singh SK. Antibiotic resistance pattern and clonal relationship of Enterobacter spp isolates from different clinical samples in Odisha, India. Blood. 4:12-15. DOI : 10.52711/0974-360X.2022.00047

18. Alexandre A, Laranjo M, Oliveira S. Natural populations of chickpea rhizobia evaluated by antibiotic resistance profiles and molecular methods. Microb Ecol. 2006; 51(1):128-136. https://doi.org/10.1007/s00248-005-0085-3

19. Caufield PW, Saxena D, Fitch D, Li Y. Population structure of plasmid-containing strains of Streptococcus mutans, a member of the human indigenous biota. J Bacteriol. 2007; 189(4):1238-1243.DOI: https://doi.org/10.1128/jb.01183-06

20. Bergmann R, Nerlich A, Chhatwal GS, Nitsche-Schmitz DP. Distribution of small native plasmids in Streptococcus pyogenes in India. Int J Med Microbiol. 2014; 304(3-4):370-378.DOI :https://doi.org/10.1016/j.ijmm.2013.12.001

21. Murchison HH, Barrett JF, Cardineau GA, Curtiss R. Transformation of Streptococcus mutans with chromosomal and shuttle plasmid (pYA629) DNAs. Infect Immun. 1986;54(2):273-282.DOI: https://doi.org/10.1128/iai.54.2.273-282.1986

22. Chansley PE, Kral TA. Transformation of fluoride resistance genes in Streptococcus mutans. Infect Immun. 1989;57(7):1968-1970. DOI: https://doi.org/10.1128/iai.57.7.1968-1970.1989

23. Lin K, Tierno Jr PM, Komisar A. Increasing antibiotic resistance of Streptococcus species in New York City. Laryngoscope. 2004; 114(7):1147-1150.DOI:https://doi.org/10.1097/00005537-200407000-00003

Received on 31.01.2022 Modified on 23.12.2022

Research J. Pharm. and Tech 2023; 16(10):4799-4804.

DOI: 10.52711/0974-360X.2023.00778