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IJPCBS 2014, 4(2), 372-377 Veena Shetty et al. ISSN: 2249-9504

INTERNATIONAL JOURNAL OF PHARMACEUTICAL, CHEMICAL AND BIOLOGICAL SCIENCES

Available online at www.ijpcbs.com Research Article

COMPARATIVE EVALUATION OF ACTIVITY OF THE ESSENTIAL PLANT OILS ON MULTIDRUG RESISTANT NOSOCOMIAL PATHOGENS Shilpa M Shenoy1, A. Veena Shetty1* and Vinaya Bhat2 1Department of Microbiology, K.S. Hegde Medical Academy, Nitte University, Mangalore - 575018, Karnataka, India. 2Department of Prosthodontics and Implantology, A.B. Shetty Memorial Institute of Dental Sciences, Nitte University, Mangalore - 575018, Karnataka, India.

ABSTRACT The problem of microbial resistance is growing and the lookout for the use of antimicrobial drugs in the future is still uncertain. Therefore, actions must be taken to reduce this problem, and to offer appropriate and efficient antimicrobial drugs to the patient. Down the ages essential oils and other of plants have evoked interest as sources of natural products. They have been screened for their potential uses as alternative remedies for the treatment of many infectious diseases. This study was undertaken to evaluate the antimicrobial activity of commercially available oil, oil, Castor oil, oil, Neem oil and extracted oreganum oil on the multidrug resistant nosocomial pathogens. The oreganum oil, clove oil eucalyptus oil and neem oil showed antimicrobial activity. With proper studies they can be a beacon of hope in long dark tunnel of dwindling era of new antibiotics.

Keywords: multidrug resistance, , nosocomial pathogens.

INTRODUCTION namely Acinetobacter species, multidrug-resistant Antimicrobial resistance has been on rise ever (MDR) P.aeruginosa, and carbapenem- resistant since the use of antibiotics. The dream to Klebsiella species and Escherichia coli—are overcome the resistance of against emerging as significant pathogens around the the is still a long way from world. The therapeutic options for these fulfilment. A report published by the Infectious pathogens are so extremely less and despite Diseases Society of America, published in 20091, ongoing efforts the number of new antibacterial stressed on the increase of the antimicrobial drugs approved for marketing in the United States resistance among the gram positive and gram and the world continues to decrease2. negative pathogens that cause infection in the Due to the adverse effects of chemical antibiotics hospitals and in the community. The report and the resistant microorganisms, the scientists identified “ESKAPE” pathogens Enterococcus have explored new sources for antimicrobial faecium, Staphylococcus aureus, Klebsiella agents especially among medicinal plants. pneumoniae, Acinetobacter baumanii, Numerous plants are known to possess medicinal Pseudomonas aeruginosa, and Enterobacter or food values for humans and are used for species to emphasize that they currently cause the treatment of human ailments3. Traditional majority of hospital infections and effectively is the main source of medical care for a “escape” the effects of antibacterial drugs. Several great proportion of the population of the highly resistant gram-negative pathogens— developing world.

372 IJPCBS 2014, 4(2), 372-377 Veena Shetty et al. ISSN: 2249-9504

placing Cefotaxime (30µg), Ceftazidime(30µg) and Essential oils and plant extracts based on Ceftriaxon(30µg) on inoculated Mueller-Hinton ethnomedicinal uses are potential sources of new agar plates and incubated for 24 hours at 37°C. antimicrobial compounds against microbial When the inhibition zones of the drugs for gram strains. The combined use of plant extracts or negative bacteria were ≤27mm for essential oils and antibiotics are useful in Cefotaxime(30µg), ≤22mm for Ceftazidime(30µg), decreasing drug resistant problems4. Plant ≤25mm for Ceftriaxon(30µg) respectively ,the products are a great choice and an alternate to strain was suspected as a potential ESBL synthetic antibiotics due to their ready producer. The potential ESBL isolate in E.coli and availability, biodegradability and low cost factor. Klebsiella pneumoniae was then confirmed using The antimicrobial property of essential oils from Cefotaxime (30µg), ceftazidime (30µg), alone and plants was used since old ages, but now there is in combination with clavulanic acid on Muller renewed interest in the search for plants with hinton agar. The test organism was identified as a anti-microbial activity. There are numerous ESBL producer when the zone of inhibition of medicinal plant species that are used by Indian ceftazidime (30µg) or Cefotaxime (30µg) population. combined with inhibitor showed difference of The emergence of antimicrobial resistance, ≥5mm compared with cephalosporins alone 5. coupled with availability of fewer new ESBL production in P.aeruginosa and A.baumanii antimicrobial drugs, led us to conduct this study was detected by double disc synergy test 6. using the plant oils which are commonly found in Synergy was determined between a disc of the Indian household. We used Coconut oil, Castor amoxyclav (20 μg amoxycillin and 10 μg clavulanic oil, Eucalyptus oil, Neem oil, Clove oil and acid) and a 30-μg disc of each 3GC test antibiotic Oreganum oil against the Extended spectrum Beta placed 15 mm apart on a lawn culture of the lactamase(ESBL) and Methicillin resistant isolate. The test organism was considered to Staphylococcus aureus (MRSA) producing produce ESBL if the zone size around the nosocomial pathogens. antibiotic disc increased towards the amoxyclav The aim of the study was to determine the disc. This increase occurs because the clavulanic antimicrobial susceptibility pattern of organisms acid present in the amoxyclav disc inactivates the producing ESBL in Gram negative bacilli and ESBL produced by the test organism MRSA obtained from the hospitalised patients. To determine the antimicrobial activity of the Antimicrobial Activity of the plant oils commercially available essential plant oils on the Five commercially available plant oils were most common ESBL and MRSA organisms isolated procured: Eucalyptus oil, Neem oil, Castor oil, from hospitalised patients. Coconut oil and Clove oil; Ooms laboratory Shimoga. Oreganum oil was extracted through MATERIALS AND METHODS standard procedures. All the oils were checked for The most common bacterial nosocomial authenticity by a pharmacognosist. The purchased pathogens E.coli, Klebsiella pneumonia, oils were not diluted and not altered chemically by Pseudomonas aeruginosa, Acinetobacter baumanii any solvent or processing. and Staphylococcus aureus were isolated from the Antibacterial activity of the oils were tested using various clinical specimens obtained from the the agar well diffusion method7. The test inoculum hospitalised patients. They were identified by (0.5 McFarland’s turbidity) was spread onto colony morphology, Gram staining and standard Muller-Hinton agar by using a sterile cotton swab. biochemical tests. Antimicrobial Susceptibility Then the wells were punched by a sterile well patterns were determined by Modified Kirby- puncture. Twenty μl of oils were added to each Bauer method on Muller Hinton agar. well and incubated at 37°C for 24 h. The presence of zones of inhibition was regarded as the Detection of MRSA and ESBL presence of antimicrobial action. The diameter of Screening for Methicillin resistant Staphylococcus zone of inhibition was measured in mm. aureus(MRSA) was done by disc diffusion method Antimicrobial activity was expressed in terms of using Cefoxitin disc(30 µg). Detection of Extended average diameter of the zones of inhibition Spectrum Beta Lactamse (ESBL) enzyme was done measured. by double disc diffusion method for screening and confirmatory tests set by CLSI guidelines. Screening for ESBL production was done by

373 IJPCBS 2014, 4(2), 372-377 Veena Shetty et al. ISSN: 2249-9504

MIC and MBC DISCUSSION The broth microdilution method was used to The emergence of resistance to conventional determine Minimum inhibition concentration antimicrobials is a serious problem that (MIC) and Minimum bactericidal (MBC). All tests physicians face. This necessitates constant were performed in Mueller-Hinton broth. Briefly, development of newer agents, which can inhibit serial doubling dilutions of the were the growth of or kill resistant organisms. The oils prepared in a 96-well microtiter plate ranging of medicinal plants have been used for treatment from 100% to 0.098%. Finally, 10 μl of bacterial of various ailments since times immemorial. suspension (106 CFU/ml) was added to each well. Numerous studies have been conducted to screen The plates were wrapped loosely with cling film to the antimicrobial activity of different plant ensure that the bacteria did not get dehydrated. extracts and essential oils against human The plates were prepared in triplicates, and then pathogens, but this study was conducted to find they were placed in an incubator at 37°C for 18-24 the antimicrobial activity of commonly found hours8. The results were expressed as mean and plant oils in every Indian household against the or ranked in order of importance as percent. multidrug resistant nosocomial pathogens. These oils are commonly used as food flavouring agents, RESULTS in small doses in clinical setting for numerous The organisms selected for the study were medical conditions such as liver and gallbladder multidrug resistant organisms. Organisms which disturbances, abscesses, headaches, appendicitis, were found to be resistant to three or more epilepsy, hemorrhoids, constipation, diarrhoea, different classes of antibiotics were defined as intestinal obstructions, skin diseases, hyper multidrug resistant organisms. All the gram activity in children and to avert threatened negative bacterial isolates included in the study abortion in pregnant women, antiparasitic 9,10,11. were resistant to third generation cephalosporin. Our investigation on commonly used plant oils But were susceptible to carbapenems, and showed that the oreganum oil had the highest Staphylococcus aureus was resistant to cefoxitin activity against the Multi Drug Resistant (MDR) but susceptible to vancomycin. organisms tested. It showed better activity than The essential plant oils exhibited very good the control antibiotics used in the study. Our antimicrobial activity on the nosocomial bacterial results are also supported by previous authors pathogens as compared to the control antibiotics, oreganum oil has shown great antimicrobial i.e Imipenem for gram negative bacteria and activity against numerous microorganisms, it has Vancomycin for S.aureus. The oreganum oil been proved to be antibacterial-not only against exhibited the most antibacterial activity against the human pathogens but also against animal, the tested bacteria. It showed better antibacterial plant pathogens and food spoiling bacteria, activity as compared to the control antibiotics. antifungal. Clove oil exhibited good antimicrobial Eucalyptus oil and clove oil also exhibited better activity against the MDR organisms in our study. antibacterial activity as compared to the neem oil, The studies conducted by L Mayaudi12, H.J.D coconut oil and castor oil. The oils exhibited the Doroman13, D Stojkovic14 found that Oregano and highest antibacterail activity against E.coli and clove oil had had highest inhibitory activity on the S.aureus as compared to other tested organisms tested pathogens. Clove oil exhibited great (Table 1). antibacterial activity against Multidrug resistant The MIC was defined as the lowest concentration Pseudomonas15 and E.coli16. A large scale study of the extract at which the does conducted by Mendell Friedman17 on 96 essential not demonstrate the visible growth. The MBC was oils and 23 compounds against the bacteria defined as the lowest concentration of the extract isolated from food and clinical cases found that at which the incubated microorganism was oregano oil and clove oil were effective against completely killed. The average of 3 values was E.coli, L.monocytogens, S.enterica, C.jejuni. calculated providing the MIC and MBC values for Similarly, another study18 found that among the the tested oils. Among the oils studied, oregano oil 56 essential oils tested exhibited highest exhibited the lowest inhibitory and bactericidal antimicrobial activity against A.baumanii, E.coli, concentration against all the microorganisms K.pneumoniae, P. aeruginosa, and S.aureus. tested. Eucalyptus oil, clove oil and neem oil Eucalyptus oil also exhibited great antimicrobial followed not far behind (Table 2). activity against the multidrug resistant organisms tested in our study. In support to our study, studies conducted by T Takahashi19 and Fatemeh

374 IJPCBS 2014, 4(2), 372-377 Veena Shetty et al. ISSN: 2249-9504

Mohsen Nezhad20 found that antimicrobial activity study did not exhibit any antimicrobial activity was found greatly among the Gram positive against the multidrug resistant nosocomial organisms including MRSA. Biljana’s21 study bacteria. In contrast to our study, the studies found strong antimicrobial activity of Eucalyptus conducted by other authors to evaluate the essential oil on S.pyogens, E.coli, Candida , antimicrobial property of castor seeds have Acinetobacter species and Klebsiella species. exhibited antimicrobial activity29. But the tests Neem an evergreen has been a source of were conducted on freshly prepared ethanolic, numerous medicinal properties. Our study methanolic aqueous extracts and freshly showed a moderate antimicrobial activity against extracted oils from the seeds. These studies show the MDR organisms. Our work is in support with that the aqueous extract was comparitvely less the study done by M M Nijire22,they found that active against the tested organisms. They found neem oil had comparatively much lesser activity that P.aeruginosa and E.feacalis were resistant to against the MDR organisms. Whereas studies the extracts as compared to other organism30. conducted by other authors found that the Further studies are required to confirm the ethanolic and dichloromethane leaf extracts of activity of the commercially available oils against Neem were more active towards the bacterial the multidrug resistant pathogens. Through our species used in their study 23. Aditi Grover.et al24 study we observed that the freshly prepared oils found that the methanol extract of Neem leaves exhibited better antibacterial activity as compared were effective as 100μg/ml Gentamicin against to the commercially available oils. This may be one Bacillus subtilis and 20μg/ml Tetracycline against of the reason as our study did not show any both Staphylococcus aureus and Proteus vulgaris antibacterial activity in coconut and Castor oils. and 10μg/ml Gentamycin against Pseudomonas Natural drugs have been a part of the evolution of aeruginosa. R K Upadhyaya25 and his group found human, healthcare for thousands of years. that among all essential oils, they tested almond Nowadays nearly 88% of the global populations and neem oils were found to be highly turn to plant derived as their first line bactericidal, as they exhibited lowest MIC and of defence for maintaining health and compacting MBC values and high growth inhibition zone diseases. One hundred and nineteen secondary diameter in comparison to antibiotics. Present plant metabolites derived from plants are used study reveals significantly higher broad-spectrum globally as drugs, 15% of all angiosperms have antibacterial activity in essential oils than been investigated chemically and of that 74% of antibiotics i.e., tetracycline, ampicillin and pharmacologically active plant derived ciprofloxacin. components were discovered 31. Plants are rich in Coconut and its numerous products find its uses in a wide variety of secondary metabolites such as our daily life, coconut oil is consumed on daily , terpenoids, alkaloids, flavonoids, etc. basis in coastal Karnataka region. Hence it was which have been found In-vitro to have medicinal used in our study, according to our results properties. Pharmacological studies have accepted commercially available coconut oil did not show the value of medicinal plants as potential source of any antimicrobial activity against the multi drug bioactive compounds32. Phyto-chemicals from resistance organisms. Our results support a study medicinal plants serve as lead compounds in conducted by Chiaw Mei Sia et.al26, they also found antimicrobial discovery33,34. This may be due to that the commercial available coconut oil did not the known botanical facts that from different exhibit any antimicrobial properties. But there are location grown on different geographical, climatic studies conducted on the fermented coconut oil 27 and zoological conditions may demonstrate which exhibited antibacterial activity against different properties. The disparity sometimes may Bacillus subtilis, Escherichia coli, Pseudomonas also be attributed to the superior equipments and fluorescence, Bacillus cereus and Salmonella. chemicals, extraction methods. Hence the need of Studies conducted on mesocarp extracts, the hour is to standardise the different extraction extracted from different solvents also exhibited procedures and identify the antimicrobial antimicrobial activity against E.coli and Salmonella component by researched method. This is very spp28. Castor oil was selected in the study due to much required to combat the headlong evolution its easy availability in every household. The of multidrug resistance in the pathogens. commercially available Castor oil used in this

375 IJPCBS 2014, 4(2), 372-377 Veena Shetty et al. ISSN: 2249-9504

Table 1: Antibacterial activity of the plant oils by well diffusion method Test material Mean Zone of Inhibition E.coli K.pneumoniae P.aeruginosa A. baumanii S. aureus 1. Oregano oil 22.17±0.75 20.11±0.66 18.03±0.87 17.9±0.55 20.11±0.0 2. Eucalyptus oil 16.50±0.83 18±0.65 16.65±0.06 15.75±0.65 18.17±0.76 3. Clove oil 15±0.63 16.50±0.45 12.08±0.78 13.05±0.98 18.16±0.85 4. Neem oil 12±0.65 13.25±0.05 12.06±0.80 10.06±0.09 15.5±0.67 5. Coconut oil 6 6 6 6 6 6. Castor oil 6 6 6 6 6

Table 2: MIC and MBC expressed by the oils by microtitre plate method Test material Organism E.coli K.pneumoniae P.aeruginosa A. baumanii S. aureus MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC 1. Oregano oil 0.048 0.097 0.097 0.781 0.195 0.781 0.195 0.781 0.048 0.097 2. Eucalyptus oil 0.097 0.390 0.390 0.781 0.390 1.562 0.390 1.562 0.097 0.781 3. Clove oil 0.097 0.390 0.781 1.562 0.781 3.125 0.781 3.125 0.390 0.781 4. Neem oil 0.781 3.125 1.562 3.125 1.562 3.125 3.125 6.250 0.781 1.562

REFERENCES Antioxidant Activities in Cassia auriculata. 1. Helen WB, George HT, John SB, John E E,Jr, Glob J Pharmacol. 2009;3(3):127-130. David G, Louis BR et al. Bad Bugs, No 8. Yu J, Lei J, Yu H et al. Chemical Drugs: No ESKAPE! An Update from the Composition and Antimicrobial Activity of Infectious Diseases Society of America. The Essential Oil of Scutellaria barbata. Clin Infect Dis. 2009;48(1):1-12. Phytochemistry. 2004;65(7):881-4. 2. Talbot GH, Bradley J, Edwards JE Jr, 9. Christopher B. Ed. The Royal Horticultural Gilbert D, Scheld M and Bartlett JG. Bad Society A-Z Encyclopedia of Garden Bugs Need Drugs: An Update on the Plants. London: Dorling Kindersley. Development Pipeline from the 1996:884–885. Antimicrobial Availability Task Force of 10. Betancur-Galvis LA, Morales GE, Forero JE the Infectious Diseases Society of and Roldan, J. Cytotoxic and Antiviral America. Clin Infect Dis. 2006;42:657–68. Activities of Colombian Medicinal Plant 3. Cowan MM. Plant Products as Extracts of the Euphorbia Genus, Antimicrobial Agents. Clin Microbiol Rev. Memórias Do Instituto Oswaldo Cruz. 1999;12(4):564. 2009;97(4):541-546. 4. Mahboubi M and Ghazian Bidgoli F. In 11. Odungbemi T. Outline and Pictures of Vitro Synergistic Efficacy of Combination Medicinal Plants from Nigeria, University of Amphotericin B with Myrtus Communis of Lagos Press, Yaba Lagos, Nigeria. Essential Oil against Clinical Isolates of Letters in Applied Microbiology. Candida albicans. Phytomedicine. 2006:283. 2010;17(10):771-4. 12. Mayaudi L, Carricajo A, Zhiri A and Aubert 5. Clinical and laboratory standards G. Comparison of Bacteriostatic and institute.2010. Performance standards for Bactericidal Activity of 13 Essential Oils antimicrobial susceptibility testing. CLSI against Strains with Varying Sensitivity to M100- S20U. Update June 2010. Clinical Antibiotics. Lett Appl Microbiol. and Laboratory Standards Institute, 2008;47(3):167-73. Wayne, PA, USA. 13. Dorman HJD and Deans SG. Antimicrobial 6. Jarlier V, Nicolas MH, Fournier G and Agents from Plants: Antibacterial Activity Philippon A. Extended Spectrum Βeta- of Plant Volatile Oils. Journal of Applied Lactamases Conferring Transferable Microbiology. 2000;88:308–316 Resistance to Newer Β- Lactam Agents in 14. Stojkovic D, Jasmina G, Ana C, Nikolic M, Enterobacteriaceae: Hospital Prevalence Ristic M, Jovana S et al. Investigation on and Susceptibility Patterns. Rev Infect Dis. Antibacterial Synergism of Origanum 1988;10:867–878. Vulgare and Thymus Vulgaris Essential 7. Anushia C, Sampathkamur P and Oils. Arch Biol Sci. Belgrade, Ramkumar L. Antibacterial and 2013;65(2):639-643.

376 IJPCBS 2014, 4(2), 372-377 Veena Shetty et al. ISSN: 2249-9504

15. Mahboobi M, Shahcheraghi F, Mehdi M 24. Aditi Grover, B.S.Bhandari and Nishant and Feizabad P. Bactericidal Effects Of Rai. Antimicrobial Activity of Medicinal Essential Oils from Clove, Lavender and Plants-Azadirachta indica A. Juss, Allium Geranium on Multi-Drug Resistant cepa L. and Aloe vera L. International Isolates Of Pseudomonas aeruginosa. Journal of Pharmtech Research. Iranian Journal Of Biotechnology. 2011;3(2):1059-1065. 2006;4:2. 25. Upadhyay RK, Dwivedi P and Ahmad S. 16. Nuñez L and Aquino MD. Microbicide Screening Of Antibacterial Activity of Six Activity of Clove Essential Oil (Eugenia Plant Essential Oils against Pathogenic caryophyllata). Brazilian Journal of Bacterial Strains. Asian Journal Of Medical Microbiology. 2012:1255-1260. Sciences. 2010;2(3):152-158. 17. Friedman M, Philip R. Henika, and 26. Sia cm, Yim hs and Lai CM. Commercial Mandrell RE. Bactericidal Activities of Virgin Coconut Oil: Assessment of Plant Essential Oils and Some of their Antimicrobial Potential. As J Food Ag-Ind. Isolated Constituents against 2010;3(06):567-579. Campylobacter Jejuni, Escherichia coli, 27. Rini Handayani, Joko Sulistyo and Rita Listeria monocytogenes, and Salmonella Dwi Rahayu. Extraction of Coconut Oil enterica. Journal of Food Protection. (Cocos nucifera L.) through Fermentation 2002;65:(10):1545–1560. System. Biodiversita. 2009;10(3):151- 18. Hammer KA, Carson CF and Riley TV. 157. Antimicrobial activity of essential oils and 28. Verma V, Bhardwaj A, Rathi S and Raja other plant extracts. Journal of Applied RB. A Potential Antimicrobial Agent from Microbiology. 1999;86:985–990. Cocos nucifera Mesocarp Extract; 19. Takahashi T, Kokubo R and Sakaino M. Development of a New Generation Antimicrobial Activities of Eucalyptus Antibiotic. ISCA J Biological Sci. Leaf Extracts and Flavonoids from 2012;1(2):48-54. Eucalyptus maculata. Letters in Applied 29. Momoh AO, Oladunmoye MK and Adebolu Microbiology. 2004;39:60–64. TT. Evaluation of The Antimicrobial and 20. Fatemoh MN, Habib Z, Ali M, Morteza S Phytochemical Properties of Oil from and Abbas Y. Antimicrobial Activity of Castor Seeds (Ricinus communis Linn) Eucalyptus Extracts on Methicillin Bull. Environ. Pharmacol Life Sci. Resistant Staphylococcus aureus. 2012;1(10):21 – 27. Research Journal of Biological Sciences. 30. Jombo GTA and Enenebeaku MNO. 2009;4(8):905-908. Antibacterial Profile of Fermented Seed 21. Damjanović-Vratnica B, Đakov T, Šuković Extracts of Ricinus communis: Findings D and Damjanović J. Antimicrobial Effect from a Preliminary Analysis. Nigerian of Essential Oil Isolated from Eucalyptus Journal of Physiological Sciences. 23(1- globulus Labill. From Montenegro Czech J. 2):55-59. Food Sci. 2011;29(3):277–284. 31. Raja RV, Ramanathan T and Savitha S. 22. Njire MM, Budambula NLM and Kiiru JN. Studies on Wound Healing Property Of Antimicrobial Effects of Selected Herbal Coastal Medicinal Plants. J Biosci Tech. Extracts on Multi-Drug Resistant Gram- 2009;1:39-44. Negative Bacterial Strains. Proceedings of 32. Biswas K, Chattopadhyay I, Banerjee RK, 2010 JKUAT Scientific Technological and and Bandyopadhyay U. Biological Industrialization Conference. 2010:131- Activities and Medicinal Properties Of 143 Neem (Azadirachta Indica). Curr Sci. 23. Rajasekaran C, Meignanam E, 2002;82:1336–1345. Vijayakumar V, Kalaivani T, Ramya S, 33. Chakravarthy BK and Gode KD. Isolation Premkumar N, Siva R and Jayakumararaj of Epicatechin From Pterocarpus R. Investigations on Antibacterial Activity Marsupium and Its Pharmacological of Leaf Extracts of Azadirachta Indica A. Action. Planta Medica. 1985;1:56-59. Juss (Meliaceae): A Traditional Medicinal 34. Ebi GC and Ofoefule SI. Antimicrobial Plant of India. Ethnobotanical Leaflets. Activity of Pterocarps osun Stems. 2008;12:1213-17. Fitoterapia. 71:433- 435.

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