1. INTRODUCTION:

Group B Streptococcus (GBS), known as Streptococcus agalactiae is a Gram-positive, haemolytic, chain-forming bacterium and a commensal within the genital tract flora in approximately 25% of healthy adult women¹. This bacteria is a cause of serious infection in newborns, pregnant women, and elderly people suffering from chronic diseases. In neonates, GBS infection most commonly causes pneumonia, meningitis, and sepsis. In addition to maternal genital colonization and neonatal infection. The bacterium can also cause urinary tract infection (UTI)². Antibiotic resistance in GBS is not widely appreciated and many laboratories do not undertake full antibiotic susceptibility tests of clinical isolates.

However, antibiotic combinations such as penicillin plus gentamicin are often used to manage severe GBS infection, exposing patients to enhanced risk of antibiotic-related toxicity and resistance of Streptococcus type B against antibiotic.

In October 1998 and March 1999, an in vitro study conducted by Argentinian multicentre indicated that beta-hemolytic Streptococci were susceptible to penicillin, chloramphenicol and ceftriaxone. A similar study showed that beta-hemolytic Streptococci including Streptococcus agalactiae are resistance towards β-lactam antibiotics such as tetracycline and microlides lactones³. S. agalactiae was already resistant to gentamicin with MIC⁴.

Bacterial infections are one of the prominent causes of health problems⁵. Drug resistance to many pathogens has been reported from different countries⁶. However, the condition is alarming in both developing and developed countries because of indiscriminate application of antibacterial agents. In this situation, it is necessary to look for new antimicrobial agents from other sources with minimal undesirable effects such as herbs⁷. Herbs have been used as a medicine long time ago and they have been known for their use to cure many kinds of diseases^8,9. Medicinal herbs contain secondary metabolites known as phyto-ingredients that can either inhibit the growth of the pathogens or kill them and have least toxicity effect to host cells¹⁰. These properties make them good alternatives to develop new antimicrobial medicine¹¹. It has been shown that the leaves of P. betle possess tremendous beneficial effects including antimicrobial, antioxidant, and anti-diabetic, wound healing and other properties^12,13. However, there is little information about the antibacterial properties of P. betle against S. agalactiae. Therefore, this study investigated the antibacterial activity of P. betle leaves against the Gram-positive bacteria S. agalactiae, and the Gram negative bacteria E-coli. The antibacterial activity was investigated using Kirby-Bauer Disc Diffusion Method¹⁴, Minimal Inhibition Concentration and Minimal Bacterial Concentration^15,16. This study also compared the effectiveness of P. betle extract as an antibacterial with the commercial antibiotics used against these two species.

2. MATERIAL AND METHODS:

2.1 Plant Extraction:

Piper betle was bought from local market in Kelantan. Fresh leaves were washed and air dried. After air dried, the P. betle leaves were pulverised by using an electric blender to form powder. Leaves powder was kept at room temperature and stored in a dark place until use. Leaves powder (2g) was mixed with 20ml of 70% ethanol in a conical flask. The flask was plugged with cotton wool and stored for 7 days at room temperature (28°C - 38°C). Seven days later, the mixture was filtered using sterile muslin cloth. The filtrate was subjected to evaporation using rotatory vacuum evaporator. The residue was freeze-dried and stored at -20°C until use¹⁷.

2.2 Bacteria inoculums preparation:

Bacteria culture was provided by aquatic animal health unit, University Putra Malaysia (UPM). Bacteria suspension was prepared to conduct antimicrobial test. Bacteria suspension was activated by adding 0.05ml of frozen stock bacteria into 10ml TSB (Oxoid, England) and incubated overnight at 37°C. The content of broth streaked into TSA (Oxoid, England) petri dishes and incubated for 24 h at 37°C. Inoculums were provided using one loopful of bacteria colony from TSA petri dishes into 10ml of TSB and incubated in the TSB liquid in an orbital shaker for 24 h at 37°C. One ml of the latest bacterial culture was transferred to a newly made TSB. The bacterial suspension was adjusted until turbidity was the same as 0.5 McFarland Standard, obtaining a final cell concentration 10⁸ CFU ml¹⁸.

2.3 Disk diffusion method to determine antimicrobial sensitivity:

Antimicrobial characteristics of the P. betle against S. agalactiae and E.coli examined by paper disk diffusion method¹⁴. Furthermore, the bacteria culture (10⁸ CFU ml) was spread on the Mueller Hinton agar petri dish before putting the paper disks on the impregnated petri dishes. Sterile 6.0mm diameter Whitman No. 1 filter paper disk seeded into the P. betle extract as follows: 50, 75 and 100mg/ml solvent. A 20µl of the extracts were used per disks and the disks were subjected to dryness before placing into the petri dishes. Oxytetracycline 20 µg/ml, streptomycin 20µg/ml, tetracycline 20µg/ml, ampicillin 20µg/ml, erythromycin 20µg/ml, and gentamicin 20µg/ml served as positive controls, whereas ethanol was employed as a negative control¹⁹. Then, the petri dishes were incubated at 30°C for 24 h. After that, the antibacterial activity was determined by measuring the diameter of the inhibition zone formed around the disk. According to Sheikhlar et al (2013), the inhibition zone bigger than 15mm was considered as strong activity, the zone from 10-15mm was considered as moderate activity, and the zone smaller than 10mm was considered as a weak activity.

2.4 Minimal Inhibitory Concentration and Minimal Bactericidal Concentration:

The minimum inhibitory concentration (MIC) was determined by broth dilution method. A 100µL of each standardised bacterial inoculums well was mixed with extract of 100%, 50% and 10% concentrations in 96 well plates. The plates were incubated at 37°C for 24h. The lowest concentration of the extract that showed no turbidity was recorded as MIC²⁰. After incubation for determining the MIC, inoculum from each well (96 well plates) streaked into Mueller Hinton agar and incubated at 37°C for 24 h. The lowest concentration that prevented bacterial growth was recorded as MBC²¹.

2.5 Statistical analysis:

Data obtained were analysed by one way ANOVA using SPSS statistics.

3. RESULTS:

3.1 Zone of Inhibition of S. agalactiae upon selected antibacterial discs and different concentration of P. betle.

The zone of inhibition of S. agalactiae upon selected antibacterial discs and different concentration of P. betle is shown in Figure 1. The enrofloxacin with concertation of 5µg showed the highest zone of inhibition (ZOI) (average diameter of 21mm) amongst other antibacterial discs. For Oxytetracycline with concertation of 30µg, the ZOI was 18mm in diameter and there was no ZOI for the antibiotic Ampicillin with concertation of 10µg which indicate that S. agalactiae is resistant to Ampicillin. Different concentrations of P. betle gave different results for area involvement as ZOI. The lowest concentration of P. betle extract (50mg/mL) showed the lowest ZOI with a width of 18.5mm in average, followed by the P. betle extract concentration of 75mg/mL which showed a 19.5mm wide ZOI in average, while at (100mg/mL) of P. betle concentration provided the highest ZOI which is 20.5mm on average (Figure 1).

Figure 1: Zone of Inhibition of S. agalactiae upon selected antibacterial discs and different concentration of P. betle.

As shown in Figure 2, E. coli ZOI towards Enrofloxacin with a concertation of 5µg showed 9mm in the first trial and 10mm in second trial with the average of 9.5mm. A concertation of 30µg from Oxytetracycline showed 8 mm ZOI on both first and second trial. No zone of inhibition was shown towards ampicillin (Figure 2). P. betle extract with a concentration of 50mg/mL showed low ZOI amongst other P. betle extracts which is 10mm in average, followed by the P. betle extract with 75mg/mL that showed 18mm in average. The third P. betle concentration showed the highest zone of inhibition which is 19.5 mm average in diameter (Figure 2).

Figure 2: Zone of inhibition of E. coli upon selected antibacterial disc at different concentrations of P. betle.

3.2 Minimal inhibition concentration (MIC) for S.agalactiae and E. coli upon P. betle extract.

The Minimal inhibition concentration (MIC) based on optical turbidity according to the mean of the negative control (TSB+Bacteria) is shown in Table 1. The MIC for the 100mg/mL P. betle used were at 3.125mg/ml of and 1.5625mg/ml. The MIC for the 75mg/mL P betle used were both at 4.6875mg/ml, whereas the MIC for the 50mg/ml P. betle used were at 12.5mg/ml and 25mg/ml. The OD of the negative control for S. agalactiae were 3.2368 and 2.339 with the mean 2.7879 (The mean value is a reference value to select the MIC).

Table: 1 Minimal inhibition concentration (MIC) for S. agalactiae and E. coli upon P. betle extract based on optical density.

Bacteria	Concentration of P. betle extract
	50 mg/ml MIC	75 mg/ml MIC	100 mg/ml MIC
E. coli (OD) upon concentration (mg/ml)	18.67	4.688	2.344
S. agalactiae (OD) upon concentration concentration (mg/ml)	1.172	0.659	0.073

First we study the effect of the P. betle extract on the gram positive strain S. agalactiae, in the low concentration of 50mg/ml the MIC was 1.172mg/ml. After increasing the concentration to 75mg/ml the MIC decrees to 0.659mg/ml. However, in the highest concentration the MIC was 0.073mg/ml. Furthermore the study of the MIC on gram negative strain E.coli with different concentration (50, 75, 100mg/ml) revealed the following results 18.67, 4.688 and 2.344 respectively (Table 1) .

4. DISCUSSION:

4.1 Zone of Inhibition of S. agalactiae upon selected antibacterial discs and different concentration of P. betle.

Group B Streptococcus is uniformly sensitive towards penicillin and ampicillin²². Yet, it was proven that GBS can be treated with beta-lactam antibiotics. In this study, S. agalactiae showed resistance toward this type of antibiotic. A case report revealed that S. agalactiae isolated from the red hybrid tilapia already resistant towards many antibiotics such as ampicillin. Though there is a great year difference between West Country to eastern country especially the south East Asian in term of selection of antibiotics for S. agalactiae infection. Literally, south East Asian are still using the antibiotic which already resistant in Malaysia. This scenario might be due to antibiotic resistance. Unethical use of antibiotic in the treatment can lead to antibiotic resistance²³. However, S. agalactiae can be treated with ampicillin²². Nevertheless, it is not practical to use ampicillin in Malaysia. Ampicillin resistance is not new in E. coli^24,25 The use of oxytetracycline in the management of S. agalactiae and E. coli should be revised; this is because oxytetracycline is the common choice of treatment and prevention in the case of S. agalactiae infection in fish. Prophylactic or treatment contributes to develop antibacterial resistance. Oxytetracycline resistant bacteria develop due to frequent use of oxytetracycline for preventing and controlling bacterial pathogen in salmon farming in china²⁶. Based on the current study results, it was revealed that resistance of both S. agalactiae and E. coli towards penicillin and oxytetracycline is well proven. Presence of the inhibition zone suggests that P. betle extract dose have antibacterial properties against S. agalactiae and E. coli. This herb extract showed a zone of inhibition higher than ampicillin 10µg and oxytetracycline 30µg. This indicates that the usage of P. betle extract in S. agalactiae and E. coli infection is more efficient than ampicillin and oxytetracycline. In this study, the ZOI created by the P. betle extract ranging from 10-21mm in diameter is similar to a previous study even though they are using different bacteria. Usually ZOI with the P. Betle ranges from 19-22mm in diameter²⁷. Results reveal that P. betle gave bigger ZOI in S.agalactiae compared to E. coli. A study on P. betle showed bigger ZOI in the case of Gram positive bacteria compared to Gram negative bacteria²⁸. Structural anatomical and physiological cell wall differentiation of Gram positive and Gram negative gave an impact on the uptake of the antibacterial drugs. Gram positive bacteria are more susceptible to the inhibitory effect because of single layer of peptidogly can and lack of the natural sieve effect against chemical, whereas gram negative bacteria are multilayered and complex cell wall structure which makes them less susceptible than gram positive bacteria²⁹. However, Kirby-Baeur method for analyzing antibacterial sensitivity is a qualitative approach in determining whether the bacteria is susceptible or resistant towards antibacterial drugs. In order to know the extract amount of antibacterial use to inhibit bacterial growth, it is important to know the minimal inhibition concentration.

4.2 Minimal inhibition concentration of S. agalactiae and E. coli upon P. betle extract:

S. agalactiae seems to be more sensitive towards P. betle leaves extract compared to E. coli. Lower concentration of P. betle seems to inhibit S. agalactiae compared to E. coli that requires more concentration to inhibit the growth. It is proved that P. betle is more sensitive towards Gram positive bacteria than Gram negative bacteria. It was shown that the MIC for S. aureus was 0.78mg/ml which is more sensitive compared to Vibrio vulnificus with 3.15mg/ml. S. agalactiae required 0.073 mg/ml to inhibit the growth, whereas S. aureus required 0.78mg/ml³⁰. Accordingly, the bacteria are more sensitive towards P. betle compared to S. aureus. However, the concentration of P. betle requires as antibacterial is different between bacteria types. Furthermore, Najiah (2012) showed that V. vulnificus required concentration of P. betle (3.15mg/ml) and this concentration is higher compared to both S. agalactiae and E. coli in the current study. Our study showed that enrofloxacin concentration required to inhibit S. agalactiae growth was 0.016mg/ml, and a concentration of 0.15mg/ml required to inhibit E. coli. Compared to P. betle extract, a concentration of 0.073mg/ml needed to inhibit S. agalactiae and 2.344mg/ml needed to inhibit E. coli. It seems that the amount of P. betle extract almost the same as enrofloxacin to treat both S. agalactiae and E. coli. This study proves that P. betle can inhibit bacterial growth on certain minimal concentration and can be an alternative medication to treat S. agalactiae and E. coli. However, in vivo study about direct application of P. betle extract to the animals with S. agalactiae and E. coli infection should be established in the future. This is important to know the efficacy of extract towards infection and body system response towards its effect. The phytochemical substances should be identified and screened to identify the active ingredients that act as antibacterial agents.

5. CONCLUSIONS:

Piper betle leaves or Sirih extracts can be considered as one of the solutions to the antibiotic resistance in the S. agalactiae and E. coli. This study showed that both S. agalactiae and E. coli were resistant towards ampicillin and oxytetracycline. On the other hand, the extract of P. betle showed ZOI like the commercial antibacterial and minimal concentration guide to known the minimal concentration to inhibit bacteria growth.

6. ACKNOWLEDGMENT:

The authors are grateful to Middle East University-Amman-Jordan for the financial support granted to cover the publication fees of this research article.

7. CONFLICT OF INTEREST STATEMENT:

The authors declare that there is no conflict of interest

8. REFERENCES:

1. Campbell JR, Hillier SL, Krohn MA, Ferrieri P, Zaleznik DF, Baker CJ. Group B streptococcal colonization and serotype-specofic immunity in pregnant women at delivery. Obstetrics and Gynecology. 2000; 96(4): 498-503

2. Tan CK, Carey AJ, Ipe D, and Ulett G C. Current understanding of streptococcal urinary tract infection. Clinical Management of Complicated Urinary Tract Infection. 2011. 51-70.

3. Lopardo H. Antimicrobial resistance in β-hemolytic Streptococcus in Argentina. Communicating current research and education topics and trends in applied microbiology. Formatex Journal. 2007; 12: 794-798.

4. Liddy H, and Holliman R. (2002). Group B Streptococcus highly resistant to gentamicin. Journal of Antimicrobial Chemotherapy. 2002; 50(1): 142-143.

5. Paul S and Saha D. Comparative Study of the Efficacy of Barleria prionitis Leaf Extracts against Bacteria. Asian J. Pharm. Res. 2012; 2(3): 107-110.

6. Jeyanthi T, Subramanian P, Kumaravel P. A Comparative Analysis of Antibacterial Activity of Withania somnifera Root Extract with Commercial Antibiotics. Asian J. Pharm. Res. 2013; 3(2): 98-102.

7. Killedar SG, Kope KI, Sangle SB, Tamboli MS. Standardization and Antimicrobial Activity of Watery Fluid at Floral Base of Spathodea campanulata (Pal). Asian J. Pharm. Ana. 2011; 1(1): 19-21.

8. Ranjan B, Honey J, Birendra S. Standardization and phytochemical investigation of Berberis aristata. Asian J. Pharm. Ana. 2012; 2(3): 81-84.

9. Hemalatha M, Arirudran B, Thenmozhi A, Mahadeva Rao US. Antimicrobial Effect of Separate Extract of Acetone, Ethyl Acetate, Methanol and Aqueous from Leaf of Milkweed (Calotropis gigantea L.). Asian J. Pharm. Res. 2011; 1(4): 102-107.

10. Ramasubramaniaraja R. Pharmacognostical Phytochemical Including GC-MS Investigation of Ethanolic Leaf Extracts of Abutilon indicum (Linn). Asian J. Pharm. Ana. 2011; 1(4): 88-92.

11. Sheikhlar A, Alimon AR, Daud HM, Saad CR, and Shanagi H. Screening of Morus alba, Citrus limon and Trigonella foenum-graecum extracts for antimicrobial properties and phytochemical compounds. Journal of Biological Sciences. 2013; 13(5): 386-392.

12. Tee LH, Luqman Chuah A. Pin KY, Abdull Rashih, Yusuf Y.A. Optimization of Spray drying process parameters of Piper betle (Sirih) leaves extract coated with maltodextrin. Journal of Chemical and Pharmaceutical Research. 2012; 4(3): 1833-1841.

13. Jaya Preethi P. Herbal Medicine for Diabetes Mellitus: A Review. Asian J. Pharm. Res. 2013; 3(2): 57-70.

14. Bauer AW, and Kirby WM. Sherris LC. and Turck M. Antibiotic susceptibility testing by a standardized single disk method. American Journal of Clinical Pathology. 1996; l45: 493-496.

15. Tiwari P. Antimicrobial Activity of Amritarishta Prepared by Traditional and Modern Methods. Asian J. Pharm. Res. 2014; 4(2): 114-116.

16. Mohite SA, Shah RR, Patel NR. Antimicrobial Activity of Leaves extracts of Jatropha curcas. Asian J. Pharm. Res. 2018; 8(1): 17-20.

17. Harikrishnan R, Balasundaram C, Kim MC, Kim JS, Han YJ, and Heo MS. Innate immune response and disease resistance in Carassius auratus by triherbal solvent extracts. Fish and Shellfish Immunology. 2009; 27(3): 508-515.

18. Sagdıç O, and Özcan M. Antibacterial activity of Turkish spice hydrosols. Food Control.2003; 14(3): 141-143.

19. Jain B, Tewari A, Bhandari BB, and Jhala MK. Antibiotic resistance and virulence genes in Streptococcus agalactiae isolated from cases of bovine subclinical mastitis. Veterinarski arhiv.2012; 82(5): 423-432.

20. Unnisa N, Tabassum H, Ali MN, and Ponia K. Evaluation of antibacterial activity of five selected fruits on bacterial wound isolates. International Journal of Pharma and Bio Sciences.2012; 3(4): 531-546.

21. Aibinu I, Adenipekun T, Adelowotan T, Ogunsanya T, and Odugbemi T. Evaluation of the antimicrobial properties of different parts of Citrus aurantifolia (lime fruit) as used locally. African Journal of Traditional. Complementary, and Alternative Medicines.2007; 4(2): 185

22. Christian H, Hofele RV, Urlaub H, and Ficner R. Insights into the activation of the helicase Prp43 by biochemical studies and structural mass spectrometry. Nucleic Acids Research.2014; 42(2): 1162-1179.

23. Stiller RJ, Padilla L, Choudary R, Tinghittella T, Laifer S. Group β Streptococcus antibiotic resistance patterns in pregnant women. Conn Med, Jun.Jul. 2003; (6): 323-6.

24. Tadesse DA, Zhao S, Tong E, Ayers S, Singh A, Bartholomew MJ, and McDermott PF. Antimicrobial drug resistance in Escherichia coli from humans and food animals, United States, 1950–2002. Emerging Infectious Diseases. 2012; 18(5): 741.

25. Muthukumaran P, Padmapriya P, Salomi S, Umamaheshwari R, Kalaiarasan P, Malarvizhi C. In Vitro Antimicrobial Activity of Leaf Powder. Asian J. Pharm. Res. 2011; 1(4): 108-110.

26. Miranda CD, and Zemelman R. Bacterial resistance to oxytetracycline in Chilean salmon farming. Aquaculture. 2002; 212(1-4): 31-47.

27. Jahir Alam Khan. JA and Naveen Kummar. Evaluation of antibacterial properties of extract of Piper betle leaf. Journal of pharmaceutical and biomedical science. 2011; 11(01).

28. Hoque MM, Rattila S, Shishir MA, Bari ML, Inatsu Y, and Kawamoto S. Antibacterial activity of ethanol extract of betel leaf (Piper betle L.) against some food borne pathogens. Bangladesh Journal of Microbiology. 2011; 28(2): 58-63.

29. Pradan SA and Biswasroy P. Golden heart of nature: Piper betle. Journal of Pharmacognosy and Phytochemistry.2013; 1 (6): 147-167.

30. Najiah M, Aqilah NI, Lee KL, Khairulbariyyah Z, Mithun S, Jalal KCA, and Nadirah M. Massive mortality associated with Streptococcus agalactiae infection in cage-cultured red hybrid tilapia Oreochromis niloticus in Como River, Kenyir Lake, Malaysia. Journal of Biological Sciences. 2012; 12(8): 438-442.

Received on 19.11.2020 Modified on 25.12.2020

Research J. Pharm. and Tech. 2021; 14(9):4920-4924.

DOI: 10.52711/0974-360X.2021.00855