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World Journal of Pharmaceutical Research SJIF Impact Factor 7.523 World Journal of Pharmaceutical Research

Pallavi et al. Volume 6, Issue 3, 193-201.

Research Article

ISSN 2277– 7105

CHLOROPHYLL AND MANUKA HONEY - SUITABLE ALTERNATE FOR VISA CAUSING POST CESAREAN WOUND INFECTION *Pallavi Jayavanth1, Getaneh A.1, Mohammedaman M.1, Ashenafi A.1 Mathan M.2, Yasmeen M.3 1

Dept of Medical Laboratory Sciences, College of Medicine and Health Sciences, Arba Minch University. Ethiopia. 2

Dept of Diagnostic and Allied Health Sciences, Faculty of Health and Life Sciences. Management and Science University. Malaysia. 3

Dept of Clinical Biochemistry. Mubarak Al Kabeer Hospital, Jabriya. Kuwait. ABSTRACT

Article Received on 13 Jan. 2017,

Antibiotic resistance and post cesarean wound infections have been a

Revised on 02 Feb. 2017, Accepted on 23 Feb. 2017

continuous problem for clinicians. Alternate low cost natural

DOI: 10.20959/wjpr20173-8021

antibacterial agents are evaluated in parallel for their efficacy due to escalating antibiotic resistance. In view of this, a study was conducted

*Corresponding Author

to record the efficacy of chlorophyll and manuka honey against

Pallavi Jayavanth

vancomycin intermediate Staphylococcus aureus (VISA). Confirmed

Dept of Medical Laboratory

VISA isolates from post cesarean wound infection patients were

Sciences, College of

collected from infection control practitioner and subjected to

Medicine and Health

antibacterial assay against different concentrations of chlorophyll and

Sciences, Arba Minch University. Ethiopia.

manuka honey as per standard procedure. Stock solutions of chlorophyll and manuka honey were prepared at 1% w/v concentration

with dilution range from 25μl/ml to 250μl/ml and subjected to minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against VISA. MIC and MBC recorded for chlorophyll and manuka honey was 150μl/ml, 100μl/ml and and MBC 200μl/ml, 150μl/ml respectively. Chlorophyll and manuka honey demonstrated promising results to use as alternate antibacterial agent against VISA causing post cesarean wound. Pharmacological preparation of chlorophyll and manuka honey embedded in bandages could aid as effective therapeutic alternate to treat post cesarean wound infection. KEYWORDS: Chlorophyll, manuka honey, drug resistance, vancomycin, VISA, post cesarean wound. www.wjpr.net

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INTRODUCTION Morbidity, mortality and extended hospital stay due to antibiotic resistance have been reported for many decades.[1-2] Acronyms, abbreviations and warning oriented terms such as ESKAPE, ESCAPE, BAD BUGS, SUPER BUGS, NIGHTMARE BACTERIA have been coined to initiate zone specific and cause specific antibiotic resistance stewardship globally. [34]

The current infectious disease statistics have approximated to that of pre antibiotic era and

bacteria developing resistance overtaking the race of new antibiotic development.[5-6] Methicillin resistant Staphylococcus aureus (MRSA) has been a dominant bug globally causing hospital and community acquired infections, vancomycin was introduced due to MRSA, which in turn resulted in reduced sensitivity to vancomycin, these strains were tagged as VISA – vancomycin intermediate S. aureus.[7-10] The first vancomycin resistance was reported in Japan 1996, followed by similar reports from other parts of the world.[11-14] Cesarean section and cesarean wound infection is still reported to be common in developed and developing countries and Staphylococcus aureus is the most isolated pathogen from post cesarean wound.[15-19] Plant biologist have reported antimicrobial efficacy of numerous plant extracts and natural products; extensive studies are in progress in search of new antimicrobial agents due to increased reports of antibiotic resistance.[20-21] Chlorophyll has been used for wound healing since long and scientifically proven to have antimicrobial properties. [22-32] Honey is the ancient natural antiseptic; many reports claim healing and antibacterial properties against spectrum of bacteria causing wound infection. [33-41] In view of this, a preliminary study was undertaken to detect the antimicrobial efficacy of chlorophyll and manuka honey against VISA causing post cesarean wound infection. MATERIALS AND METHODS Bacterial Strain: VISA was obtained from Infection Control Practitioner. Malaysia. Chlorophyll: 100% liquid chlorophyll was purchased locally from Natural Health Farm Limited Malaysia. Manuka honey: Commercially prepared manuka honey MG 100+ was purchased from Summer Pacific Limited Malaysia.  Preparation of manuka honey and chlorophyll stock solution: Chlorophyll and manuka honey stock solution was prepared separately by adding 2.5g of chlorophyll and manuka honey to 250ml of water and stirred to dissolve.  www.wjpr.net

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Preparation of inoculum: The standard bacterial strain of VISA was inoculated in sterile nutrient broth and was incubated at 370C for 24 hours. The turbidity of the broth was matched with McFarland’s standard solution to obtain a bacterial inoculum of 10 8 (colony forming unit) CFU/ml. Minimal Inhibitory Concentration (MIC): The MIC is defined as the lowest concentration of chlorophyll and manuka honey that is able to inhibit the growth of VISA. A series of 10 test tubes were prepared. Each test tube was added with 5ml of Muller Hinton broth and calibrated with 100μl of bacterial suspension. Different concentrations of chlorophyll and manuka honey ranging from 25μl/ml to 250μl/ml was added to each test tube. The wide dilution range was considered to compare the efficacy. The tubes were incubated at 370C for 24h. After incubation, turbidity of each tube was visually inspected. Clear test tube indicated break point.[42] Minimal Bactericidal Concentration (MBC): Suspension from selected incubated test tubes was inoculated on nutrient agar plate using a sterile cotton swab. The plates were incubated at 370C for 24hours, bacteriostatic and bactericidal indicators were recorded. RESULTS Table 1 - Minimal Inhibitory Concentration of Chlorophyll and Manuka Honey against VISA Minimal Inhibitory Concentration (μl) 25 50 75 100 125 150 175 200 Chlorophyll + + + + + + + Manuka Honey + + +  (+) indicates turbid test tubes; (-) indicates clear test tubes.  Sample

225 +

250 +

Table 2 - Minimal Bacterial Concentration of Chlorophyll and Manuka Honey against VISA Sample Chlorophyll Manuka Honey 

25 + +

50 + +

Minimal Bacterial Concentration (μl) 75 100 125 150 175 200 + + + + + + + + + +

225 + +

250 + +

(+) indicates turbid test tubes; (-) indicates clear test tubes. 

The MIC and MBC of chlorophyll and manuka honey against VISA was 150 μl/ml, 100

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μl/ml and 200 μl/ml,150 μl/ml respectively. Chlorophyll exhibited moderate sensitivity while honey exhibited high sensitivity comparatively as indicated in Table 1 & 2. DISCUSSION Decades ago the efficacy of the chlorophyll and honey was studied and documented. In practice context, insignificant data explains the dominance of newer antibiotics. This study reveals the use of chlorophyll and manuka honey as topical agent for post cesearan wound infection which can also be used for other surgical site infections. The chlorophyll efficacy recorded in the current study is similar with other reports.[24,25,28] Chlorophyll sensitive VISA was the prominent feature of the study. Xuhui (2015) reported VISA from a patient who never received vancomycin treatment, this explains the intriguing antibiotic resistance factors due to the van gene responsible for the generation of resistance.[43-45] The efficacy of manuka honey recorded in this study is in accord with other reports, although it was dismissed as a worthless but harmless substance.[46-52] In contrast, a report in 2016 explained therapeutic manuka honey is no longer so alternate.[53] However, in this study manuka honey was found to be effective against VISA. Revamil medical grade honey has demonstrated good broad spectrum antibacterial activity and also as good skin disinfectant.[54] In addition, Honey was found to exhibit good synergism with antibiotics.[55-57] Antimicrobial efficacy of the different brands of chlorophyll and honey will help to address the concern of using natural products for wound healing, as some reports claim the overuse of topical antibiotics resulted in antibiotic resistance.[58,59,60] CONCLUSION Multiple drug resistance has disturbing statistics warning complete layover of antibiotics if precautions in different therapeutic spheres are not strictly exercised. For many decades, natural antimicrobial agents have been continuously screened and many of them are commercialized with no statistics of their actual use for treatment. Chlorophyll and manuka honey efficacy has been reported with exceptional antimicrobial activity and wound healing properties. Pharmacological preparation of chlorophyll and manuka honey embedded separately in bandages as wound healing patch or PC (post cesarean) patch would prove advantageous. In addition, a study on pharmacological synergism of chlorophyll and manuka honey will help reevaluate the antibacterial potency. ACKNOWLEDGEMENT We thank the Authorities of the Management and Science University, Shah Alam, Selangor, www.wjpr.net

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Malaysia for extending requested facilities in commencement and completion of this research and Dr. Yasmeen Muneer for the support and advice. We do not have any conflicts of interest to declare. REFERENCES 1. Sharma A. ―Antimicrobial resistance: no action today, no cure tomorrow‖. Indian Journal of Medical Microbiology, 2011; 29(2): 91–92. 2. Cohen ML. ―Changing patterns of infectious disease‖. Nature, 2000; 406(6797): 762–767. 3. Boucher HW, Talbot GH, J. S. Bradley JS. ―Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America‖ Clinical Infectious Diseases, 2009; 48(1): 1–12.  4. Peterson LR. ―Bad bugs, no drugs: no ESCAPE revisited,‖ Clinical Infectious Diseases, 2009; 49(6): 992–993. 5. Arias CA, Murray BE. ―Antibiotic-resistant bugs in the 21st century—a clinical superchallenge,‖ The New England Journal of Medicine, 2009; 360(5): 439–443. 6. Basakand SM, Rajurkar N. ―Newer𝛽-lactamases and E.coli— a cause of concern,‖ Trends in Infectious Diseases, S. K. Saxena, Ed., 2014. chapter 3, pp. 47–72, Intech, Rijeka, Croatia, 2014. 7. Susana G, Alexander T. Mechanisms of vancomycin resistance in Staphylococcus aureus. The Journal of Clinical Investigation, 2014; 124(7): 2836 – 2840. 8. Howden BP, Davies J.K, Johnson PD, Stinear TP. & Grayson, ML. Reduced vancomycin susceptibility in Staphylococcus aureus, including vancomycin-intermediate and heterogeneous vancomycin-intermediate strains: resistance mechanisms, laboratory detection and clinical implications. Clin Microbiol, 2010; Rev 23: 99–139. 9. Charles PG, Ward PB, Johnson P.D, Howden B.P. & Grayson ML. Clinical features associated

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