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

RESEARCH ARTICLE In Vitro Inhibition of Staphylococcus aureus subsp. aureus (ATCC 6538TM) by Artemether-Lumefantrine Tablets: A Comparative Study of Three Dosage Strengths Opoku Solomon and Nyanor Isaac Department of Quality Control, Entrance Pharmaceuticals and Research Centre, No. 16 Okpoi Gonno, Spintex Road, Post Office Box CT 10805, Accra, Ghana Sickle Pan Africa Research Consortium, Kumasi Centre for Sickle Cell Disease, Komfo Anokye Teaching Hospital, Kumasi, Ghana


INTRODUCTION
Staphylococcus aureus is both a human pathogen and a commensal [1]. It colonizes about 30 percent of human population [2]. S. aureus is the most pathogenic species of the genus Staphylococcus and it is implicated in both community-acquired and nosocomial infections [3]. 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 [4].
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 [3]. The major factor for the success of S. aureus as a pathogen is its notable capacity to acquire antibiotic resistance [5]. There have been reports of emergence of S. aureus strains that are resistant to the following antibiotics: oxacillin, vancomycin, Mupirocin and Clindamycin [6].
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 [1]. S. aureus is a major pathogen in Africa and other developing countries [7]. In Sub-Saharan Africa and other tropical areas, it frequently causes invasive diseases [8]. Studies done in Ghana revealed that S. aureus is the third most frequently isolated microorganism from patients [9] and the second most prevalent bacterium among patients from teaching, regional and district hospitals and has a multi-drug resistant rate of 42.3% [10].
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 [11].
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 [12] 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 [13]. 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 [14]. 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 [15]. Oral formulations of artemether-lumefantrine are available as tablet and dispersible formulations and have similar pharmacokinetic properties [16]. Studies have reported antimicrobial activities of artemisinin and its derivatives against a range of pathogenic bacteria including Staphylococcus aureus [17,18].
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.

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 4 o C on Mueller-Hinton agar slants (HiMedia Laboratories Private Limited, Mumbai, India) using seed-lot culture maintenance technique with three passages [19]. 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.

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]. 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].

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).

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

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.

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 10 8 CFU/mL [6]. The suspension was then diluted in sterile physiological saline to a concentration of 1 x 10 7 CFU/mL.

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 10 4 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 35 o C for 20 hours.

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].

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 10 8 CFU/mL [6]. The inoculum was diluted in Mueller-Hinton broth to a final concentration of 5 x 10 5 CFU/mL.

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].

Incubation of Macrodilution Tubes
The inoculated macrodilution tubes were incubated aerobically at 35 o C for 20 hours.

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]. A test was considered valid when definite turbidity occurred in growth-control tube. In instances where growth turbidity in the tubes weredifficult 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.
These are footnote texts for Tables 1 and 2. Should be cut and pasted at the appropriate places.

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.

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