The Open Microbiology Journal




ISSN: 1874-2858 ― Volume 13, 2019
RESEARCH ARTICLE

In Vitro Inhibition of Staphylococcus aureus subsp. aureus (ATCC® 6538™) by Artemether-Lumefantrine Tablets: A Comparative Study of Three Dosage Strengths



Opoku Solomon1, *, Nyanor Isaac2
1 Department of Quality Control, Entrance Pharmaceuticals and Research Centre, No. 16 Okpoi Gonno, Spintex Road, Post Office Box CT 10805, Accra, Ghana
2 Sickle Pan Africa Research Consortium, Kumasi Centre for Sickle Cell Disease, Komfo Anokye Teaching Hospital, Kumasi, Ghana

Abstract

Purpose:

Antibiotics are progressively failing in the fight against infections due to S. aureus because the bacterium has an outstanding ability to acquire multi-antibiotic resistance and become resistant to most antibiotics. Multi-drug resistant S. aureus poses a major threat to the foundation upon which standard antibacterial chemotherapy stands, hence the need to consider non-antibiotic solutions to manage invasive bacterial infections. This study investigated the inhibitory activities of three dosage strengths of artemether-lumefantrine tablets against Staphylococcus aureus subsp. aureus (ATCC® 6538™) and determined the minimum concentrations of the tablets that are able to completely inhibit growth of the bacterium in vitro.

Methods:

The agar dilution and broth macrodilution techniques were used to determine the susceptibility of the Staphylococcus aureus subsp. aureus (ATCC® 6538™) strain to artemether-lumefantrine 20/120mg, 40/240mg and 80/480mg tablets.

Results:

The most active inhibitor was artemether-lumefantrine 80/480mg tablet with a minimum inhibitory concentration value of 2.5mg/mL while artemether-lumefantrine 20/120mg and 40/240mg tablets exhibited moderate but equal activities against the test strain.

Conclusions:

The study has revealed that artemether-lumefantrine, an antimalarial drug, also has anti-staphylococcal properties and inhibits S. aureus in vitro. This study presents the first report on the in vitro activity of artemether-lumefantrine tablet against S. aureus and suggests the need to consider it as an alternative in the treatment of staphylococcus infections.

Keywords: Minimum inhibitory concentrations, Multi-antibiotic resistance, Artemether-lumefantrine, Test strain, S. aureus, staphylococcus infections.


Article Information


Identifiers and Pagination:

Year: 2018
Volume: 12
First Page: 397
Last Page: 403
Publisher Id: TOMICROJ-12-397
DOI: 10.2174/1874285801812010397

Article History:

Received Date: 30/8/2018
Revision Received Date: 6/12/2018
Acceptance Date: 10/12/2018
Electronic publication date: 31/12/2018
Collection year: 2018

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© 2018 Solomon and Isaac.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


* Address correspondence to this author at the Department of Quality Control, Entrance Pharmaceuticals and Research Centre, No. 16 Okpoi Gonno, Spintex Road, Post Office Box CT 10805, Accra, Ghana; Tel: +233205090159; E-mail: slmnopoku@gmail.com




1. INTRODUCTION

Staphylococcus aureus is both a human pathogen and a commensal [1Tong SY, Davis JS, Eichenberger E, Holland TL, Fowler VG Jr. Staphylococcus aureus infections: Epidemiology, pathophysiology, clinical manifestations, and management. Clin Microbiol Rev 2015; 28(3): 603-61.[http://dx.doi.org/10.1128/CMR.00134-14] [PMID: 26016486] ]. It colonizes about 30 percent of human population [2Stark L. 2013.Staphylococcus aureus: Aspects of pathogenesis and molecular epidemiology. Linköping: Linköping University Electronic Press. Linköping University Medical Dissertations, 1371.: Department of Clinical and Experimental Medicine, Linköping University.]. S. aureus is the most pathogenic species of the genus Staphylococcus and it is implicated in both community-acquired and nosocomial infections [3Costa AR, Batistão DWF, Ribas RM, Sousa AM, Pereira O, Botelho CM. “Staphylococcus aureus virulence factors and disease,” in Microbial Pathogens and Strategies for Combating Them: Science, Technology and Education 2013; 702-10.]. S. aureus has been reported as the causative agent of wide variety of diseases and infections such as boil, wound infection, pustule, subcutaneous and sub-mucosa abscesses, osteomyelitis, mastitis, impetigo, septicemia, meningitis, bronchopneumonia, food poisoning and urinary tract infections [4Nsofor CA, Nwokenkwo VN, Ohale CU. Prevalence and antibiotic susceptibility pattern of staphylococcus aureus isolated from various clinical specimens in South East Nigeria. MOJ Cell Sci Rep 2016; 3(2): 00054.[http://dx.doi.org/10.15406/mojcsr.2016.03.00054] ].

S. aureus infections are often exceptionally difficult to treat because of the large population heterogeneity, phenotypic switching, intra-strain diversity, hypermutability and the small colony variants [3Costa AR, Batistão DWF, Ribas RM, Sousa AM, Pereira O, Botelho CM. “Staphylococcus aureus virulence factors and disease,” in Microbial Pathogens and Strategies for Combating Them: Science, Technology and Education 2013; 702-10.]. The major factor for the success of S. aureus as a pathogen is its notable capacity to acquire antibiotic resistance [5Watkins RR, David MZ, Salata RA. Current concepts on the virulence mechanisms of meticillin-resistant Staphylococcus aureus. J Med Microbiol 2012; 61(Pt 9): 1179-93.[http://dx.doi.org/10.1099/jmm.0.043513-0] [PMID: 22745137] ]. There have been reports of emergence of S. aureus strains that are resistant to the following antibiotics: oxacillin, vancomycin, Mupirocin and Clindamycin [6 Clinical and Laboratory Standards Institute. (2015). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard, 10th ed. CLSI document M07-A10. Clinical and Laboratory Standards Institute, Wayne, PA.].

In developed countries worldwide, S. aureus is the commonest cause of bacteremia and infective endocarditis and is associated with excess mortality relative to other pathogens [1Tong SY, Davis JS, Eichenberger E, Holland TL, Fowler VG Jr. Staphylococcus aureus infections: Epidemiology, pathophysiology, clinical manifestations, and management. Clin Microbiol Rev 2015; 28(3): 603-61.[http://dx.doi.org/10.1128/CMR.00134-14] [PMID: 26016486] ]. S. aureus is a major pathogen in Africa and other developing countries [7Ruffing U, Alabi A, Kazimoto T, et al. Community-associated staphylococcus aureus from Sub-Saharan Africa and Germany: A cross-sectional geographic correlation study. Sci Rep 2017; 7(1): 154.[http://dx.doi.org/10.1038/s41598-017-00214-8] [PMID: 28273954] ]. In Sub-Saharan Africa and other tropical areas, it frequently causes invasive diseases [8Magill AJ, Hill DR, Solomon T, Ryan ET. Hunter’s tropical medicine and emerging infectious diseases 9th ed. 9th ed.2013; Vol. XXIV: 1190.]. Studies done in Ghana revealed that S. aureus is the third most frequently isolated microorganism from patients [9Opintan JA, Newman MJ, Arhin RE, Donkor ES, Gyansa-Lutterodt M, Mills-Pappoe W. Laboratory-based nationwide surveillance of antimicrobial resistance in Ghana. Infect Drug Resist 2015; 8: 379-89.[http://dx.doi.org/10.2147/IDR.S88725] [PMID: 26604806] ] and the second most prevalent bacterium among patients from teaching, regional and district hospitals and has a multi-drug resistant rate of 42.3% [10Newman MJ, Frimpong E, Donkor ES, Opintan JA, Asamoah-Adu A. Resistance to antimicrobial drugs in Ghana. Infect Drug Resist 2011; 4: 215-20.[PMID: 22259250] ].

The emergence of Multi-Drug Resistant (MDR) microbial pathogens poses a major threat to the foundation upon which standard antibacterial chemotherapy stands hence the need to consider non-antibiotic solutions to manage invasive bacterial infections [11Opal SM. Non-antibiotic treatments for bacterial diseases in an era of progressive antibiotic resistance. Crit Care 2016; 20(1): 397.[http://dx.doi.org/10.1186/s13054-016-1549-1] [PMID: 27978847] ].

Antibiotics are progressively failing in the fight against infections due to S. aureus because the organism has an outstanding ability to acquire multi-antibiotic resistance [12Hiramatsu K, Katayama Y, Matsuo M, et al. Multi-drug-resistant Staphylococcus aureus and future chemotherapy. J Infect Chemother 2014; 20(10): 593-601.[http://dx.doi.org/10.1016/j.jiac.2014.08.001] [PMID: 25172776] ] and become resistant to most antibiotics. This is a serious threat to global public health and requires stakeholders to come up with a harmonized set of approaches to fight antimicrobial resistance in a multifaceted manner [13Roca I, Akova M, Baquero F, et al. The global threat of antimicrobial resistance: Science for intervention. New Microbes New Infect 2015; 6: 22-9.[http://dx.doi.org/10.1016/j.nmni.2015.02.007] [PMID: 26029375] ]. As part of the efforts to develop approaches to fight antimicrobial resistance, this study sought to investigate the inhibitory effects of a non-antibiotic agent such as artemether-lumefantrine against S. aureus.

Artemether-lumefantrine is an artemisinin-based combination therapy approved for treatment of un-complicated malaria [14Stover KR, King ST, Robinson J. Artemether-lumefantrine: An option for malaria. Ann Pharmacother 2012; 46(4): 567-77.[http://dx.doi.org/10.1345/aph.1Q539] [PMID: 22496476] ]. Artemisinin derivatives are endorsed internationally for treatment of malaria because of their high potency, rapid onset of action, broad malaria stage specificity and favourable safety profile [15Piscitelli SC, Gallicano KD. Interactions among drugs for HIV and opportunistic infections. N Engl J Med 2001; 344(13): 984-96.[http://dx.doi.org/10.1056/NEJM200103293441307] [PMID: 11274626] ]. Oral formulations of artemether-lumefantrine are available as tablet and dispersible formulations and have similar pharmacokinetic properties [16Abdulla S, Sagara I. Dispersible formulation of artemether/lumefantrine: Specifically developed for infants and young children. Malar J 2009; 8(Suppl. 1): S7.[http://dx.doi.org/10.1186/1475-2875-8-S1-S7] [PMID: 19818174] ]. Studies have reported antimicrobial activities of artemisinin and its derivatives against a range of pathogenic bacteria including Staphylococcus aureus [17Tajehmiri A, Issapour F, Moslem MN, Lakeh MT, Kolavani MH. In vitro antimicrobial activity of Artemisia annua leaf extracts against pathogenic bacteria. Adv Stud Biol 2014; 6: 93-7.[http://dx.doi.org/10.12988/asb.2014.4525] , 18Appalasamy S, Lo KY, Ch’ng SJ, Nornadia K, Othman AS, Chan LK. Antimicrobial activity of artemisinin and precursor derived from in vitro plantlets of Artemisia annua L. BioMed Res Int 2014; 2014: 215872.[http://dx.doi.org/10.1155/2014/215872] [PMID: 24575401] ].

This study examined the inhibitory activities of three dosage strengths of artemether-lumefantrine tablets against Staphylococcus aureus subsp. aureus (ATCC® 6538™) and determined their Minimum Inhibitory Concentrations (MICs) using the agar dilution and broth macrodilution methods.

2. METHODS

2.1. Bacterial Strain

The Staphylococcus aureus subsp. aureus (ATCC® 6538™) strain studied was obtained from Microbiologics Inc, St. Cloud, Minnesota USA. It was a lyophilized organism and first passage from reference strain. The strain was verified and confirmed in accordance with supplier’s protocol and the certificate of analysis. The organism was maintained at 4oC on Mueller-Hinton agar slants (HiMedia Laboratories Private Limited, Mumbai, India) using seed-lot culture maintenance technique with three passages [19Clontz L. (1998). Microbial Limit and Bioburden Tests: Validation approaches and global requirements, Interpharm Press, Inc., Buffalo Grove.]. Prior to testing, the strain was subcultured onto Mueller-Hinton agar and incubated at 35°C for three days to ensure the viability and purity of the inoculum.

2.2. Culture Media

The Mueller-Hinton agar and Mueller-Hinton broth used for the study were obtained from HiMedia Laboratories Private Limited, Mumbai, India. Each batch of the Mueller-Hinton agar was confirmed for growth promotion and Minimum Inhibitory Concentration (MIC) performance according to Clinical and Laboratory Standards Institute (CLSI) procedures [20 Clinical and laboratory standards institute. (2006). Protocols for evaluating dehydrated mueller-hinton agar; approved standard - 2nd ed. CLSI document M06-A2. Clinical and laboratory standards institute, Wayne, PA.]. The growth promotion and minimum inhibitory concentration performance characteristics of each batch of the Mueller-Hinton broth were confirmed using standard set of quality control microorganisms recommended by CLSI [21 Clinical and laboratory standards institute. (2016). Performance standards for antimicrobial susceptibility testing; 26th ed. CLSI M100S. Clinical and laboratory standards institute, Wayne, PA.].

2.3. Artemether-Lumefantrine Samples

The artemether-lumefantrine tablet samples used in this study were manufactured by Entrance Pharmaceuticals and Research Centre, a pharmaceutical manufacturing industry located in Accra, Ghana. The three artemether-lumefantrine tablet dosage strengths studied were 20/120mg, 40/240mg and 80/480mg. The excipients used in the tablet formulations were microcrystalline cellulose, aerosol, crosscarmellose sodium, polysorbate 80, talcum and magnesium stearate. These excipients were the same for all the different tablet strengths. The average masses of the tablets studied were 308mg, 582mg and 685mg for 20/120mg, 40/240mg and 80/480mg respectively. The ages of artemether-lumefantrine 20/120mg, 40/240mg and 80/480mg tablets at the time of the study were 8 months, 16 months and 8 months respectively. The tablets were aseptically ground into fine powder and prepared as stock solutions. Two-fold serial dilutions were then performed to obtain a concentration range of 0.04 mg/mL to 160 mg/mL (Tables 1 and 2).

Table 1
Scheme for preparing dilutions of artemether-lumefantrine used in agar dilution susceptibility tests.


Table 2
Scheme for preparing dilutions of artemether-lumefantrine used in broth dilution susceptibility tests.


2.4. Antimicrobial Susceptibility Testing

The agar dilution and broth macrodilution techniques described by CLSI (CLSI, 2015) were used to determine the susceptibility of the Staphylococcus aureus subsp. aureus (ATCC® 6538™) strain to the artemether-lumefantrine samples.

2.5. Agar Dilution Method

2.5.1. Preparation of Agar Dilution Plates

Artemether-lumefantrine tablet suspensions were prepared by making successive 1:2, 1:4 and 1:8 dilutions to produce a concentration range of 0.4 mg/mL to 1600 mg/mL (Table 1). One part (1.3mL) of each dilution was added to nine parts (11.7mL) of molten Mueller-Hinton agar that has been allowed to equilibrate in a water bath to 45°C. This produced a concentration range of 0.04 mg/mL to 160 mg/mL (Table 1). Growth-control plates were prepared using sterilized distilled water in place of the artemether-lumefantrine suspensions. The tubes were thoroughly mixed and poured into 90mm diameter Petri plates to result in an agar depth of 4 mm. The agar plates were allowed to solidify at room temperature.

2.5.2. Inoculum Preparation

Inoculum was prepared by making saline suspension of colonies of Staphylococcus aureussubsp. aureus (ATCC® 6538™) selected from 24-hour Mueller-Hinton agar plate. The turbidity of the bacterial suspension was adjusted to 0.5 McFarland standard which is equivalent to 1 x 108 CFU/mL [6 Clinical and Laboratory Standards Institute. (2015). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard, 10th ed. CLSI document M07-A10. Clinical and Laboratory Standards Institute, Wayne, PA.]. The suspension was then diluted in sterile physiological saline to a concentration of 1 x 107 CFU/mL.

2.5.3. Inoculation and Incubation of Agar Dilution Plates

Each agar dilution plate was inoculated with thirty-six spots of the inoculum suspension using a thirty-six pin inoculum replicator. The replicator delivered 2µL of the inoculum suspension per spot to produce a final concentration of 104 CFU/spot. Growth-control plates were inoculated before and after the inoculation of the agar dilution plates to ensure that there was no contamination or significant antimicrobial carryover during the inoculation. The inoculated plates were allowed to stand for 20 minutes at room temperature for the moisture in the inoculum spots to be absorbed into the agar. The plates were inverted and incubated at 35oC for 20 hours.

2.5.4. Determination Agar Dilution End Points

The agar plates were placed on a dark nonreflecting surface and observed with a hand lens. The minimum inhibitory concentration was recorded as the lowest concentration of artemether-lumefantrine that completely inhibited growth, disregarding a single colony or a faint haze caused by the inoculum [6 Clinical and Laboratory Standards Institute. (2015). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard, 10th ed. CLSI document M07-A10. Clinical and Laboratory Standards Institute, Wayne, PA.].

2.6. Broth Macrodilution Method

2.6.1. Inoculum Preparation

Inoculum was prepared by making saline suspension of colonies of Staphylococcus aureussubsp. aureus (ATCC® 6538™) selected from 24-hour Mueller-Hinton agar plate. The turbidity of the bacterial suspension was adjusted to 0.5 McFarland standard which is equivalent to 1 x 108 CFU/mL [6 Clinical and Laboratory Standards Institute. (2015). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard, 10th ed. CLSI document M07-A10. Clinical and Laboratory Standards Institute, Wayne, PA.]. The inoculum was diluted in Mueller-Hinton broth to a final concentration of 5 x 105 CFU/mL.

2.6.2. Preparation of Artemether-Lumefantrine Dilutions and Inoculation of Macrodilution Tubes

Stock solution of artemether-lumefantrine tablet of concentration 320 mg/mL was prepared. Intermediate solutions were prepared from the stock solution by making successive 1:2, 1:4, and 1:8 dilutions using the dilution format shown in Table 2. Then, 0.5mL of the standardized inoculum was added to each macrodilution and growth control tubes to obtain a concentration range of 0.04 mg/mL to 160 mg/mL (Table 2). Purity check of the inoculum suspension was performed by subculturing aliquots onto a Mueller-Hinton agar plate for simultaneous incubation [6 Clinical and Laboratory Standards Institute. (2015). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard, 10th ed. CLSI document M07-A10. Clinical and Laboratory Standards Institute, Wayne, PA.].

2.6.3. Incubation of Macrodilution Tubes

The inoculated macrodilution tubes were incubated aerobically at 35oC for 20 hours.

2.6.4. Determination Broth Macrodilution End Points

The minimum inhibitory concentration was read as the lowest concentration of artemether-lumefantrine that completely inhibited the growth of S. aureus in the tubes detected by unaided eye [6 Clinical and Laboratory Standards Institute. (2015). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard, 10th ed. CLSI document M07-A10. Clinical and Laboratory Standards Institute, Wayne, PA.]. A test was considered valid when definite turbidity occurred in growth-control tube. In instances where growth turbidity in the tubes were difficult to detect with unaided eye, due to higher concentrations of the powder particles, the macrodilution tube contents were cultured on Mueller-Hinton agar and observed for bacterial growth.

3. RESULTS

The three samples of artemether-lumefantrine tablets showed varying degrees of antibacterial activity against the assayed S. aureus strain and the inhibition was generally dependent upon tablet strength and concentration of tablet powder.

3.1. Agar Dilution Method

Out of 13 different concentrations of artemether-lumefantrine 80/480mg tablet tested against the S. aureus strain, 7 (53.8%) inhibited growth of S. aureus completely while 6 (46.2%) failed to inhibit S. aureus growth (Table 3). The proportions of artemether-lumefantrine 40/240mg tablet concentrations that completely inhibited S. aureus growth was 15.4%. Artemether-lumefantrine 20/120mg tablet also produced two (15.4%) inhibitory concentrations. Artemether-lumefantrine 40/240mg and 20/120mg tablets had the same MIC value of 80 mg/mL. The most active inhibitor was artemether-lumefantrine 80/480mg tablet which had an MIC value of 2.5mg/mL (Table 3).

Table 3
In vitro antimicrobial activity of artemether-lumefantrine tablets against Staphylococcus aureus subsp. aureus (ATCC® 6538™).


3.2. Broth Macrodilution Method

Seven (53.8%) out of the 13 concentrations of artemether-lumefantrine 80/480mg tablet completely inhibited growth of S. aureus while 6 (46.2%) did not inhibit S. aureus growth completely (Table 3). The proportion of artemether-lumefantrine 40/240mg tablet concentrations that completely inhibited S. aureus growth was 7.7%. Only one of the artemether-lumefantrine 20/120mg tablet concentrations inhibited growth of the test strain completely. Artemether-lumefantrine 40/240mg and 20/120mg tablets had the same MIC value of 160 mg/mL. Artemether-lumefantrine 80/480mg tablet was the most active inhibitor with an MIC of 2.5mg/mL (Table 3).

4. DISCUSSION

In this study, all the three artemether-lumefantrine tablet samples exhibited antimicrobial activity against S. aureus. This is an indication of the presence of a common anti-staphylococcal agent in all three tablet samples. The ability of the artemether-lumefantrine tablets to inhibit S. aureus growth support the findings of some studies which reported that artemisinin derivatives have the potential to inhibit growth of S. aureus (Tajehmiri et al., 2014, Appalasamy et al, 2014). The antimicrobial activity of the tablets may also be due to the lumefantrine components or the excipients or synergistic effect of some or all the compounds in the formulation hence the need for further studies in order to pinpoint the S. aureus inhibitor(s) in artemether-lumefantrine tablet.

Artemether-lumefantrine 80/480mg tablets, having the highest strength, recorded the least MIC (2.5 mg/mL), making it the most efficacious among the three tablets studied in the in vitro inhibitions of S. aureus. The MIC, the degree of inhibition and trend of activity of artemether-lumefantrine 80/480mg tablet against the S. aureus strain did not differ in both agar dilution and broth macrodilution methods. For artemether-lumefantrine 40/240mg and 20/120mg tablets, MICs were higher using the broth macrodilution method when compared to the agar dilution method. This finding is comparable to the one reported by Benning and Mathers in a similar study on veterinary antibiotics against Clostridium perfringens strains originating from porcine and avian sources [22Benning VR, Mathers JJ. Comparison of agar dilution and broth microdilution methods of anaerobic antimicrobial susceptibility testing using several veterinary antibiotics against Clostridium perfringens strains originating from porcine and avian sources. Veterinary Anaerobes and Diseaeses 1999; 5: 561-9.].

Although artemether-lumefantrine 40/240mg tablet has twice the strength of artemether-lumefantrine 20/120mg tablet, interestingly, both tablets had same MIC value in the agar dilution method (80 mg/mL) and in the broth macrodilution method (160 mg/mL). This could be due to an intrinsic property of artemether-lumefantrine tablets that makes tablets with strengths of 40/240mg and below express antimicrobial activity that does not depend on tablet strength but rather on the concentration of the tablet suspension. This observation might also be as a result of the differences in the ages of the tablets. The artemether-lumefantrine 40/240mg tablet (16 months) were relatively older than the 20/120mg tablets (8 months). Since the Active Pharmaceutical Ingredients (APIs) of the drug combination has the ability to degrade over time [23Hall Z, Allan EL, van Schalkwyk DA, van Wyk A, Kaur H. Degradation of artemisinin-based combination therapies under tropical conditions. Am J Trop Med Hyg 2016; 94(5): 993-1001.[http://dx.doi.org/10.4269/ajtmh.15-0665] [PMID: 26951346] ], the artemether-lumefantrine 40/240mg tablets might have lost some API through aging and dropped to bactericidal threshold concentrations equivalent to that of the artemether-lumefantrine 20/120mg tablets. Further studies are necessary in order to understand this phenomenon fully.

This study presents the first report on anti-staphylococcal activities of artemether-lumefantrine tablets and has shown that the tablets exhibit fairly good antimicrobial activity against Staphylococcus aureus subsp. aureus (ATCC® 6538™) in vitro.

CONCLUSION

This study has shown that artemether-lumefantrine, an antimalarial drug, has anti-staphylococcal properties and inhibits S. aureus in vitro. Artemether-lumefantrine 80/480 mg tablet, which exhibited the highest inhibitory effects against the S. aureus strain, can be studied further and considered as an alternative in the treatment of staphylococcus infections. Further studies are invited to ascertain the molecular and genetic bases of this inhibition and to explain the mechanism of action of artemether-lumefantrine in inhibiting Staphylococcus aureus.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Not applicable.

HUMAN AND ANIMAL RIGHTS

No animals/humans were used for studies that are the basis of this research.

CONSENT FOR PUBLICATION

Not applicable.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

Declared none.

REFERENCES

[1] Tong SY, Davis JS, Eichenberger E, Holland TL, Fowler VG Jr. Staphylococcus aureus infections: Epidemiology, pathophysiology, clinical manifestations, and management. Clin Microbiol Rev 2015; 28(3): 603-61.[http://dx.doi.org/10.1128/CMR.00134-14] [PMID: 26016486]
[2] Stark L. 2013.Staphylococcus aureus: Aspects of pathogenesis and molecular epidemiology. Linköping: Linköping University Electronic Press. Linköping University Medical Dissertations, 1371.: Department of Clinical and Experimental Medicine, Linköping University.
[3] Costa AR, Batistão DWF, Ribas RM, Sousa AM, Pereira O, Botelho CM. “Staphylococcus aureus virulence factors and disease,” in Microbial Pathogens and Strategies for Combating Them: Science, Technology and Education 2013; 702-10.
[4] Nsofor CA, Nwokenkwo VN, Ohale CU. Prevalence and antibiotic susceptibility pattern of staphylococcus aureus isolated from various clinical specimens in South East Nigeria. MOJ Cell Sci Rep 2016; 3(2): 00054.[http://dx.doi.org/10.15406/mojcsr.2016.03.00054]
[5] Watkins RR, David MZ, Salata RA. Current concepts on the virulence mechanisms of meticillin-resistant Staphylococcus aureus. J Med Microbiol 2012; 61(Pt 9): 1179-93.[http://dx.doi.org/10.1099/jmm.0.043513-0] [PMID: 22745137]
[6] Clinical and Laboratory Standards Institute. (2015). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard, 10th ed. CLSI document M07-A10. Clinical and Laboratory Standards Institute, Wayne, PA.
[7] Ruffing U, Alabi A, Kazimoto T, et al. Community-associated staphylococcus aureus from Sub-Saharan Africa and Germany: A cross-sectional geographic correlation study. Sci Rep 2017; 7(1): 154.[http://dx.doi.org/10.1038/s41598-017-00214-8] [PMID: 28273954]
[8] Magill AJ, Hill DR, Solomon T, Ryan ET. Hunter’s tropical medicine and emerging infectious diseases 9th ed. 9th ed.2013; Vol. XXIV: 1190.
[9] Opintan JA, Newman MJ, Arhin RE, Donkor ES, Gyansa-Lutterodt M, Mills-Pappoe W. Laboratory-based nationwide surveillance of antimicrobial resistance in Ghana. Infect Drug Resist 2015; 8: 379-89.[http://dx.doi.org/10.2147/IDR.S88725] [PMID: 26604806]
[10] Newman MJ, Frimpong E, Donkor ES, Opintan JA, Asamoah-Adu A. Resistance to antimicrobial drugs in Ghana. Infect Drug Resist 2011; 4: 215-20.[PMID: 22259250]
[11] Opal SM. Non-antibiotic treatments for bacterial diseases in an era of progressive antibiotic resistance. Crit Care 2016; 20(1): 397.[http://dx.doi.org/10.1186/s13054-016-1549-1] [PMID: 27978847]
[12] Hiramatsu K, Katayama Y, Matsuo M, et al. Multi-drug-resistant Staphylococcus aureus and future chemotherapy. J Infect Chemother 2014; 20(10): 593-601.[http://dx.doi.org/10.1016/j.jiac.2014.08.001] [PMID: 25172776]
[13] Roca I, Akova M, Baquero F, et al. The global threat of antimicrobial resistance: Science for intervention. New Microbes New Infect 2015; 6: 22-9.[http://dx.doi.org/10.1016/j.nmni.2015.02.007] [PMID: 26029375]
[14] Stover KR, King ST, Robinson J. Artemether-lumefantrine: An option for malaria. Ann Pharmacother 2012; 46(4): 567-77.[http://dx.doi.org/10.1345/aph.1Q539] [PMID: 22496476]
[15] Piscitelli SC, Gallicano KD. Interactions among drugs for HIV and opportunistic infections. N Engl J Med 2001; 344(13): 984-96.[http://dx.doi.org/10.1056/NEJM200103293441307] [PMID: 11274626]
[16] Abdulla S, Sagara I. Dispersible formulation of artemether/lumefantrine: Specifically developed for infants and young children. Malar J 2009; 8(Suppl. 1): S7.[http://dx.doi.org/10.1186/1475-2875-8-S1-S7] [PMID: 19818174]
[17] Tajehmiri A, Issapour F, Moslem MN, Lakeh MT, Kolavani MH. In vitro antimicrobial activity of Artemisia annua leaf extracts against pathogenic bacteria. Adv Stud Biol 2014; 6: 93-7.[http://dx.doi.org/10.12988/asb.2014.4525]
[18] Appalasamy S, Lo KY, Ch’ng SJ, Nornadia K, Othman AS, Chan LK. Antimicrobial activity of artemisinin and precursor derived from in vitro plantlets of Artemisia annua L. BioMed Res Int 2014; 2014: 215872.[http://dx.doi.org/10.1155/2014/215872] [PMID: 24575401]
[19] Clontz L. (1998). Microbial Limit and Bioburden Tests: Validation approaches and global requirements, Interpharm Press, Inc., Buffalo Grove.
[20] Clinical and laboratory standards institute. (2006). Protocols for evaluating dehydrated mueller-hinton agar; approved standard - 2nd ed. CLSI document M06-A2. Clinical and laboratory standards institute, Wayne, PA.
[21] Clinical and laboratory standards institute. (2016). Performance standards for antimicrobial susceptibility testing; 26th ed. CLSI M100S. Clinical and laboratory standards institute, Wayne, PA.
[22] Benning VR, Mathers JJ. Comparison of agar dilution and broth microdilution methods of anaerobic antimicrobial susceptibility testing using several veterinary antibiotics against Clostridium perfringens strains originating from porcine and avian sources. Veterinary Anaerobes and Diseaeses 1999; 5: 561-9.
[23] Hall Z, Allan EL, van Schalkwyk DA, van Wyk A, Kaur H. Degradation of artemisinin-based combination therapies under tropical conditions. Am J Trop Med Hyg 2016; 94(5): 993-1001.[http://dx.doi.org/10.4269/ajtmh.15-0665] [PMID: 26951346]

Endorsements



"Open access will revolutionize 21st century knowledge work and accelerate the diffusion of ideas and evidence that support just in time learning and the evolution of thinking in a number of disciplines."


Daniel Pesut
(Indiana University School of Nursing, USA)

"It is important that students and researchers from all over the world can have easy access to relevant, high-standard and timely scientific information. This is exactly what Open Access Journals provide and this is the reason why I support this endeavor."


Jacques Descotes
(Centre Antipoison-Centre de Pharmacovigilance, France)

"Publishing research articles is the key for future scientific progress. Open Access publishing is therefore of utmost importance for wider dissemination of information, and will help serving the best interest of the scientific community."


Patrice Talaga
(UCB S.A., Belgium)

"Open access journals are a novel concept in the medical literature. They offer accessible information to a wide variety of individuals, including physicians, medical students, clinical investigators, and the general public. They are an outstanding source of medical and scientific information."


Jeffrey M. Weinberg
(St. Luke's-Roosevelt Hospital Center, USA)

"Open access journals are extremely useful for graduate students, investigators and all other interested persons to read important scientific articles and subscribe scientific journals. Indeed, the research articles span a wide range of area and of high quality. This is specially a must for researchers belonging to institutions with limited library facility and funding to subscribe scientific journals."


Debomoy K. Lahiri
(Indiana University School of Medicine, USA)

"Open access journals represent a major break-through in publishing. They provide easy access to the latest research on a wide variety of issues. Relevant and timely articles are made available in a fraction of the time taken by more conventional publishers. Articles are of uniformly high quality and written by the world's leading authorities."


Robert Looney
(Naval Postgraduate School, USA)

"Open access journals have transformed the way scientific data is published and disseminated: particularly, whilst ensuring a high quality standard and transparency in the editorial process, they have increased the access to the scientific literature by those researchers that have limited library support or that are working on small budgets."


Richard Reithinger
(Westat, USA)

"Not only do open access journals greatly improve the access to high quality information for scientists in the developing world, it also provides extra exposure for our papers."


J. Ferwerda
(University of Oxford, UK)

"Open Access 'Chemistry' Journals allow the dissemination of knowledge at your finger tips without paying for the scientific content."


Sean L. Kitson
(Almac Sciences, Northern Ireland)

"In principle, all scientific journals should have open access, as should be science itself. Open access journals are very helpful for students, researchers and the general public including people from institutions which do not have library or cannot afford to subscribe scientific journals. The articles are high standard and cover a wide area."


Hubert Wolterbeek
(Delft University of Technology, The Netherlands)

"The widest possible diffusion of information is critical for the advancement of science. In this perspective, open access journals are instrumental in fostering researches and achievements."


Alessandro Laviano
(Sapienza - University of Rome, Italy)

"Open access journals are very useful for all scientists as they can have quick information in the different fields of science."


Philippe Hernigou
(Paris University, France)

"There are many scientists who can not afford the rather expensive subscriptions to scientific journals. Open access journals offer a good alternative for free access to good quality scientific information."


Fidel Toldrá
(Instituto de Agroquimica y Tecnologia de Alimentos, Spain)

"Open access journals have become a fundamental tool for students, researchers, patients and the general public. Many people from institutions which do not have library or cannot afford to subscribe scientific journals benefit of them on a daily basis. The articles are among the best and cover most scientific areas."


M. Bendandi
(University Clinic of Navarre, Spain)

"These journals provide researchers with a platform for rapid, open access scientific communication. The articles are of high quality and broad scope."


Peter Chiba
(University of Vienna, Austria)

"Open access journals are probably one of the most important contributions to promote and diffuse science worldwide."


Jaime Sampaio
(University of Trás-os-Montes e Alto Douro, Portugal)

"Open access journals make up a new and rather revolutionary way to scientific publication. This option opens several quite interesting possibilities to disseminate openly and freely new knowledge and even to facilitate interpersonal communication among scientists."


Eduardo A. Castro
(INIFTA, Argentina)

"Open access journals are freely available online throughout the world, for you to read, download, copy, distribute, and use. The articles published in the open access journals are high quality and cover a wide range of fields."


Kenji Hashimoto
(Chiba University, Japan)

"Open Access journals offer an innovative and efficient way of publication for academics and professionals in a wide range of disciplines. The papers published are of high quality after rigorous peer review and they are Indexed in: major international databases. I read Open Access journals to keep abreast of the recent development in my field of study."


Daniel Shek
(Chinese University of Hong Kong, Hong Kong)

"It is a modern trend for publishers to establish open access journals. Researchers, faculty members, and students will be greatly benefited by the new journals of Bentham Science Publishers Ltd. in this category."


Jih Ru Hwu
(National Central University, Taiwan)


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