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ANTIBACTERIAL ACTIVITIES OF EXTRACTS AGAINST METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS (MRSA)

George Antepim Pesewu1*, Maxwell Kelvin Billah2, Christian Adofo1, Douglas Kwang1, Michael Adetokunbo Olu-Taiwo1 and Samuel Osei-Djarbeng3

1Department of Medical Laboratory Sciences (MEDLAB), School of Biomedical and Allied Health Sciences, College of Health Sciences, University of Ghana, P. O. Box KB 143, Korle-Bu, Accra, Ghana, W/A.

2Department of Biology and Conservation Science (DABCS), School of Biological Sciences, University of Ghana, P. O. Box LG. 67, Legon, Accra, Ghana, W/A.

3Department of Pharmaceutical Sciences, Kumasi Polytechnic, P. O. Box 854, Kumasi, Ghana, W/A.

*Corresponding author: Dr. George Antepim Pesewu, Department of Medical Laboratory Sciences (MEDLAB), School of Biomedical and Allied Health Sciences, College of Health Sciences, University of Ghana, P. O. Box KB 143, Korle-Bu, Accra, Ghana, W/A

Tel: +233-277301300

Fax: +233-302-688291

E-mail: [email protected]; [email protected]; [email protected]

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ABSTRACT:

The use of and their products have over the years played enormous roles and served as efficient sources of new pharmaceuticals. The study evaluated the antibacterial activities of various solvent extracts of two species of against methicillin-sensitive and -resistant Staphylococcus aureus (MSSA and MRSA). Millipedes collected were categorized into 3 groups (Group 1 = Ophistreptus guineensis S. alone, Group 2 = Pachybolus ligulatus V. alone, Group 3 = mixture of the two species), and their products extracted using chloroform, ethanol, and distilled water. Dry extracts of the millipedes were tested against the bacteria using agar-well and paper-disc diffusion assays. Minimum inhibitory and bactericidal concentrations (MIC and MBC) of the potent solvent extracts were also determined. The highest mean diameter of zones of inhibition (29 ± 0.07 mm) and (24 ± 0.15) mm against MSSA and MRSA were observed for the ethanol extracts of Group 3 (mixture of O. guineensis + P. ligulatus) by the agar well diffusion assay. MIC and MBC values ranging from 1.56-6.25 mg/ml and 6.25-25.0 mg/ml, for the ethanol extract of the Group 3 millipedes against MSSA and MRSA were found respectively. This study has shown that extracts of millipedes possess antibacterial substances that can be effective against MRSA in-vitro.

Key words: Millipedes, extracts, zones, inhibition, MIC, MBC

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PBP2a6. Penicillin-binding proteins are cell-membrane- INTRODUCTION bound enzymes that have an important role in the bacterial Millipedes are myriapodous , with approximately cell wall synthesis, and are also the sites of targets for all β- 12,000 named species in 140 families1. The longest species lactam antibiotics7. is the Giant African millipede ( gigas) of Another mechanism of resistance in MRSA is due to the the family . Millipedes have a segmented development of drug efflux pumps in the bacteria8. The body, short antennae and many legs (Plates 1a, b). Most process involves the production of efflux-related proteins in body segments have two pairs of legs that stick out from the the bacteria which export an intensive range of antibiotics sides of the body (each segment is really 2 segments fused from the cell, resulting in reduced steady-state levels of together). The second-to-last segment has no legs and the antimicrobials in the bacteria cell, and therefore increases last segment, known as the anal segment, serves as the exit resistance or reduced susceptibility9. It has been established point for waste. Millipedes breathe through spiracles, holes that QacA and NorA, which are multidrug transporters in positioned in pairs along the body. The top of each segment MRSA, are responsible for the efflux of quaternary is called the tergite (tergum); the underside (between the ammonium salts, fluoroquinolones, macrolides, and pairs of legs) is called the sternite or sternum2. Millipedes tetracyclines from the bacteria10. These efflux-related hatch from eggs, with hatchlings possessing only the first proteins also actively export a wide range of structurally three pairs of legs. There is some maternal care of the eggs. dissimilar drugs from MRSA9. Millipedes molt (shed their old exoskeleton) as they grow and with each molt, they gain more segments, and legs. In the past decade, rates of MRSA infections have escalated Birds, badgers, and shrews eat millipedes. When they are in worldwide. MRSA is a major cause of nosocomial 11 danger, millipedes curl up in spiral shapes (Plate 1c), infections in UK hospitals . MRSA infections account for a protecting their soft undersides. They can also spray a bad- fifth of all hospital-acquired infections, costing the UK smelling liquid (hydrocyanic acid) that repels many National Health Service approximately £1 billion per year. predators. The problem has been aggravated by the rapid spread and high incidence of MRSA in intensive-care units11. MRSA is Antibiotic resistance has increased greatly in recent years a worldwide problem, as the bacteria have been isolated and is posing ever-increasing therapeutic problems from different continents, including India and Africa. In worldwide. The ways that bacteria overcome drug actions Ghana, the children’s block of the Korle-Bu Teaching are varied, ranging from intrinsic to acquired resistance Hospital (KBTH) in Accra was closed down in January 3 properties . Staphylococcus aureus is a Gram-positive 12-13 2012 as a result of an MRSA outbreak in the ward . The bacterium that grows both under aerobic and anaerobic continuing rise in MRSA infection rates and endemicity conditions. The organism is most common in nasal worldwide has led to calls for action to control infections membranes and skin of warm-blooded animals as a and develop anti-MRSA agents and vaccines14. However, commensal, but it is able to cause many kinds of infections, the difficulty in the treatment of MRSA has prompted such as skin infections, food poisoning, pneumonia, sepsis, scientist to search all avenues for a potent antibacterial and many others4. Methicillin-resistant Staphylococcus agent(s) against the bacteria. Scientist began with plants, aureus (MRSA) is a biotype of the S. aureus species that is and now on animals, especially amphibians, insects, and resistant to methicillin and other antibiotics that are arthropods. In Asia, centipedes are used extensively in the normally used to treat infections caused by this group of treatment of various bacterial infections and diseases15. It is bacteria5. Resistance to methicillin and other related therefore prudent that the antibacterial activity of millipedes antibiotics of the penicillin class is due to the presence of in Africa is investigated due to the difference in climate and chromosomal mecA gene, which specifies the production of species of organisms in both Asia and Africa. Therefore the an abnormal penicillin-binding protein called PBP2’ or

O.R.A. | 3 | AMERICANIJ Volume 2 2015 Issue 10 OCT-NOV AIJCSR AIJCSR-477 ISSN 2349-4425 www.americanij.com study evaluated the antibacterial properties of extracts of to soak for 48 h with intermittent agitation. After 48 h, the millipedes against standard reference control MRSA strains. mixtures were agitated vigorously for 10 min and allowed to settle for 5 min. The supernatant was then passed through MATERIALS AND METHODS Whatman No.1 filter paper (Whatman International Limited, England) to remove any solid materials. ethanol (99%) and Collection and processing of millipedes chloroform (99%) extracts were left open at room Before the start of the project, ethical clearance (No. SAHS- temperature under a ceiling fan for the solvent to evaporate ET./. 10351173/AA/28A/2013-2014) was obtained from the from the prepared extract. The absolute ethanol and Ethics and Protocol Review Committee, School of chloroform evaporated leaving the dried solid extracts at the Biomedical and Allied Health Sciences (SBAHS), College bottom of the containers. For the ethanol (70%), the solvents of Health Sciences, University of Ghana. Then live were removed from the extracts in vacuo at 37°C using a millipedes were collected from the University of Ghana Buchi Rotavapor rotary evaporator (Rose Scientific Limited, Botanical Gardens, Legon, Accra. The millipedes (Plates 1 Canada), while water in the aqueous extracts were removed and 2) were placed in sterile containers, transported to the by the use of Modulyo freeze dryer (Thermo Fisher Department of Animal Biology and Conservation Science Scientific, USA). Dry weights of the various extracts (DABCS), School of Biological Sciences, University of obtained were weighed and the percentage yields of dry Ghana and were identified as Ophistreptus guineensis extracts (%w/w) calculated using the formula below; (Diplopoda: Spirostreptidae) and Pachybolus ligulatus (Diplopoda: Pachybolidae) by the second author (Dr. 풘풆풊품풉풕 풐풇풅풓풚 풆풙풕풓풂풄풕 × ퟏퟎퟎ% Maxwell K. Billah). The millipedes were later transported to 풘풆풊품풉풕 풐풇품풓풐풖풏풅 풑풐풘풅풆풓 the Microbiology Laboratory (ML), SBAHS, Korle-Bu, Accra for the analysis. At ML samples of millipedes were Preparation of stock solutions of the millipede divided into 3 groups, (Group 1 = O. guineensis alone, extracts Group 2 = P. ligulatus alone, Group 3 = mixture of O. Chloroform and ethanol extracts of the millipedes were first guineensis + P. ligulatus). The millipedes were killed, cut dissolved in dimethyl-sulphoxide (DMSO), while aqueous into pieces, dried under sunlight for 3-4 days and crushed extracts were dissolved in distilled H2O prior to use. into powder using a grinding stone. The powders were weighed and placed in air-tight containers to avoid the Test bacteria and quality control absorption of moisture. Voucher specimens of the Reference control strains of bacteria were obtained from the millipedes were placed at the DABCS museum, School of American Type Culture Collection (ATCC), Rockville MD, Biological Sciences, University of Ghana for future USA. The test bacteria included MRSA: ATCC 43300 and reference. methicillin-sensitive Staphylococcus aureus (MSSA: ATCC 25923). The identity of the test control bacteria were Extraction from the powdered millipedes confirmed by incubating on blood agar (BA) plates, at 37°C Four solvents were employed in the extraction of the for 24 h and subjecting the test control bacteria to powdered millipedes including ethanol (99 and 70%), biochemical testing such as the catalase and coagulase tests. chloroform (99%), and distilled water (H O). For example, 2 After that the test control bacteria were then streaked on the 55 g each of the dry ground powder of O. guineensis (Group surface of Mueller-Hinton agar (MHA) plates, subjected to 1) was placed in 4 different containers, and 200 ml of cefoxitin (30 µg) discs and incubated at 37°C for 24 h, ethanol (99%), chloroform (99%), ethanol (70%), and according to the methods of Clinical Laboratory Standards distilled H O were added to each of the containers to form 2 Institute16. Zones of inhibition were measured following separate mixtures of the 4 solvents. This step was repeated overnight incubation, and compared with that of CLSI16 for Groups 2 (P. ligulatus alone) and Group 3 ( mixture of standards. O. guineensis + P. ligulatus). The preparations were allowed O.R.A. | 4 | AMERICANIJ Volume 2 2015 Issue 10 OCT-NOV AIJCSR AIJCSR-477 ISSN 2349-4425 www.americanij.com

Preparation of inocula inoculating pins. Positive controls were set up in both agar A minimum of 3-5 colonies of the test bacteria from well and paper disc diffusion assays using known overnight pure growth cultures were transferred into 5 ml of commercial antimicrobial agents including, ciprofloxacin (5 sterile physiological saline (0.85% NaCl), incubated for 2 h µg), gentamicin (10 µg), erythromycin (15 µg), tetracycline at 37°C, and the optical density determined using (30 µg), and vancomycin (30 µg) obtained from Axiom spectrophotometer (Aurora Instruments Limited, Canada) at Laboratories, India. Negative controls were also set up using 500 nm. The values obtained were then compared with the sterile distilled H2O. All the plates with the wells and the British Society for Antimicrobial Chemotherapy (BSAC) paper discs were left on the bench for 15 min to allow standards17. diffusion of the agents, and then incubated at 37°C for 24 h. After overnight incubation, the diameters of zones of Preparation of paper-discs inhibition were measured using vernier calipers. The tests Paper-discs used in the study were prepared using a were repeated 3 more times and the mean values calculated. modification of the method by Cheesbrough18. Paper-discs (6 mm diameter) were punched out from sheets of Whatman Determination of minimum inhibitory and No. 1 filter papers, using a perforator and placed in Petri bactericidal concentrations (MIC and MBC) dishes allowing a distance of 2-4 cm between each disc. In this assay, 2-3 colonies each of MSSA and MRSA were Petri dishes containing the discs were covered, sealed with first inoculated into separate universal bottles containing 10 cellophane tape, and sterilized in an autoclave at 121°C for ml sterile Mueller-Hinton broth (MHB) each, and incubated 15 min. After allowing the discs to cool to room overnight at 37C. Using ultra-violet (UV) temperature, 20 µl of the millipede extracts were pipetted spectrophotometer, the absorbance of the bacterial onto the different paper-discs using micropipette with sterile suspensions were measured (viability of each strain) and tips. The extracts were added in small quantities onto the based on CLSI16 and BSAC17 standards, the necessary paper discs and allowed to air dry before use. dilutions to obtain a final concentration of 106 colony- Evaluation of the antibacterial activity of the forming units per milliliter (CFU/ml), were calculated and the bacterial suspensions prepared accordingly using serial millipede extracts doubling dilutions method.

Agar- well and paper-disc diffusion assays The ethanol (99%) extract of Group 3 (a mixture of O. A sterile cotton swab was dipped into the standardized guineensis + P. ligulatus) showed the highest mean diameter inoculum suspension and pressed firmly on the inside wall of zones of inhibition against MSSA and MRSA used in the of the tube above the fluid level to get rid of excess present study and hence was selected for MIC and MBC inoculum on the swab. A dried surface of MHA plate was studies in 96-well microtitre plate. First, columns 1-12 of the inoculated by streaking the swab over the entire agar surface microtitre plate were marked, and using an Eppendorf in 3 directions, rotating the plate approximately 60° each pipette with sterile tips, sterile MHB (100 l) were time to ensure an even distribution of the inoculum. For the distributed from the first well to the 96-wells in the agar well diffusion assay, a sterile cork borer with a microtitre tray. Then 100 l volumes of the ethanol (99%) diameter of 6 mm was used to create equidistant wells in extract of Group 3 (a mixture of O. guineensis + P. each agar plate. With the aid of a micropipette with ligulatus) were added to wells A-H of column 1. Using a sterilized tips, 100 µl volume of each solvent millipede multichannel micropipette with 8 sterile tips attached to extract was dispensed into each of the wells. However, for each channel, the preparations in wells A-H of column 1 the paper-discs diffusion assay, laboratory prepared air dried were mixed well and 100 l of each (from column 1) paper discs with the millipede extracts were placed on the transferred to wells of column 2. The contents of column 2 surfaces of inoculated MHA plates with the aid of sterile were also thoroughly mixed and the above process repeated

O.R.A. | 5 | AMERICANIJ Volume 2 2015 Issue 10 OCT-NOV AIJCSR AIJCSR-477 ISSN 2349-4425 www.americanij.com up to wells A-H of column 11. No Group 3 (a mixture of O. O. guineensis alone showed considerable activity with mean guineensis + P. ligulatus) extracts were added to the wells in diameters of zones of inhibition of 25 ± 0.13 mm and 21 ± A-H of column 12, which served as positive growth 0.05 mm against MSSA and MRSA. The lowest mean controls. Then 100 µl of logarithmic phase cultures of the diameters of zones of inhibition were obtained for the all the bacterial suspension (106 CFU/ml) prepared above were aqueous extracts of the millipedes investigated (Table 2). added to each well in rows A-F, while 100 l of sterile Also, no activities were observed for all the extracts of MHB were added to each of the wells in rows G and H, Group 2 (P. ligulatus alone). The mean diameter of zones of which served as negative growth controls. To prevent inhibition of ciprofloxacin (5 µg) that was used as a control dehydration, the microtitre plate was covered with a sterile antibiotic ranged from 26-28 ± 0.00 mm against MSSA and plastic cover and incubated at 37C for 22-24 h. After MRSA respectively (Table 2). overnight incubation, the MIC was taken as the lowest For the paper-disc diffusion assay, the ethanol (99%) extract concentration of the ethanol (99%) millipede extract that of Group 3 (mixture of O. guineensis + P. ligulatus) showed prevented growth of the test bacteria. To determine the the highest mean diameters of zones of inhibition (15 ± 0.32 MBC of the millipede extract, 20 µl each of all the contents mm and 12 ± 0.07 mm) against MSSA and MRSA, in the microtitre tray were sub-cultured onto prepared MHA respectively (Table 3). No observable activities were found plates and incubated for 24-48 h. After incubation, the MBC for all the extracts of Group 2 (P. ligulatus alone). With the was taken as the lowest concentration of the ethanol (99%) control antibiotics, ciprofloxacin (5 µg), gentamicin (10 µg), millipede extract that prevented visible bacterial growth on erythromycin (15 µg), tetracycline (30 µg), and vancomycin the MHA plates. Controls were set up using ciprofloxacin, (30 µg), the mean diameters of zones of inhibition ranged and all the tests were performed in duplicates. from 21-28 ± 0.03 mm and 12-28 ± 0.01 mm against MSSA Statistical analysis and MRSA (Table 3).

Data obtained from the experiments were entered into a MIC and MBC determination Microsoft Excel and analyzed statistically, using ANOVA MIC and MBC values of the ethanol (99%) extract of Group and other descriptive statistics such as means and 3(a mixture of O. guineensis + P. ligulatus) are presented in percentages. Table 4. MIC and MBC values ranged from 1.56-6.25 RESULTS mg/ml and 6.25-25.0 mg/ml for the ethanol (99%) extract of the millipedes were found against MSSA and MRSA. Extracts However, for ciprofloxacin which was used as the standard control antibiotic, the MIC and MBC values ranged from Percentage (%) yields of the millipede extracts are presented 0.00019-0.00076 mg/ml and 0.391-1.563 mg/ml against in Table 1. The % yields ranged from 0.50-8.66%, with the MSSA and MRSA (Table 4). highest % yield obtained for the ethanol (99%) extracts of Group 2 (P. ligulatus alone) as presented in Table 1. The DISCUSSION lowest % yields were obtained for all the aqueous extracts of Antibacterial substances (ABS) including lysozyme, the two species of millipedes investigated. cecropin, attacin, etc have been previously found in groups 19 Antibacterial activity of arthropods and other invertebrates . It is in view of these that the extracts of O. guineensis and P. ligulatus were The highest mean diameters of zones of inhibition (29 ± investigated against MRSA, which has become a problem 0.07 mm and 24 ± 0.15 mm) against MSSA and MRSA worldwide. Ethanol (99%) extracts of Group 3 (mixture of were observed for the ethanol (99%) extract of Group 3 O. guineensis + P. ligulatus) showed the highest mean (mixture of O. guineensis + P. ligulatus) by the agar-well diameter of zones of inhibition for both agar-well and paper- diffusion assay (Table 2). Also the ethanol (70%) extracts of disc diffusion assays, even though all the extracts of Group O.R.A. | 6 | AMERICANIJ Volume 2 2015 Issue 10 OCT-NOV AIJCSR AIJCSR-477 ISSN 2349-4425 www.americanij.com

2 (P. ligulatus alone) didn’t show any observable activities study (Table 4). Therefore it can be said that ciprofloxacin in this study (Table 2). This is the very first report of the will be more effective than the ethanol (99%) extract of the antibacterial activities of extracts of O. guineensis, a mixture mixture of O. guineensis + P. ligulatus since it will require of O. guineensis + P. ligulatus against MSSA and MRSA. very small concentrations to inhibit, as well as kill the test But it must be noted that 2 compounds from the segmental bacteria. It must however be noted that the millipede secretions of benzoquinones-producing millipedes including extracts were in the crude form as compared to the standard Anadenobolus monilicornis von P. (Diplopoda: controls, and may have much higher or lower antibacterial Rhinocricidae) and Orthoporus dorsovittatus V. (Diplopoda: activity if purified. Spirostreptidae) have been reported to elicit a reaction that deters biting of mosquitoes and other insects in monkeys20- CONCLUSION 23. Antifungal activities of defensive secretions of certain From the study, extracts of O. guineensis and the mixture of millipedes of the family: Xystodesmidae including Cherokia O. guineensis + P. ligulatus have shown some level of Georgiana georgiana B., Cleptoria rileyi B., Euryurus antibacterial activities against MSSA and MRSA, even maculatus K., and Oxidus gracilis K. have been reported in though extracts of P. ligulatus alone did not show any the U.S.A.24. Also anti-sickling and antibacterial activities of considerable antibacterial activity against the test bacteria. extracts from Tachypodoiulus sp. (Diplopoda, Arthropoda) Work is on-going to isolate, purify, and identify the have been reported in Congo25. bioactive agent(s) in the millipede extracts using high performance liquid chromatography (HPLC) and other From the study, the ethanol (99%) extract of Group 3 analytical techniques. (mixture of O. guineensis + P. ligulatus) produced mean diameter of zones of inhibition that were comparable to ACKNOWLEDGEMENTS those of the standard control antibiotics, including We thank Miss Kathleen Kokor Glover, Department of erythromycin (15 µg), tetracycline (30 µg), and vancomycin Medical Laboratory Sciences (MEDLAB), School of (30 µg) as presented in Table. However, the MIC and MBC Biomedical and Allied Health Sciences (SBAHS), College values obtained for the ethanol (99%) extract of Group 3 of Health Sciences, University of Ghana during the (mixture of O. guineensis + P. ligulatus) against MSSA and antibacterial assays. MRSA were much higher as compared to the ciprofloxacin which was used as the standard antibiotic control with MIC CONFLICT OF INTEREST: None and MBC values ranging from 0.00019-1.563 mg/ml in the

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Table 1. Dry weights (g) and percentage (%) yields of the various groups of millipede extracts.

Groups Solvents Dry weights Percentage yield(s) 1 Ethanol (99%) 1.23 2.23 1 Chloroform (99%) 1.50 2.70 1 Ethanol (70%) 1.72 3.12 1 Aqueous 1.14 2.10 2 Ethanol (99%) 2.50 4.50 2 Chloroform (99%) 1.43 2.60 2 Ethanol (70%) 1.21 2.20 2 Aqueous 1.05 1.90 3 Ethanol (99%) 2.32 4.20 3 Chloroform (99%) 1.10 2.00 3 Ethanol (70%) 2.34 4.30 3 Aqueous 1.10 2.00

Group 1,=O. guineensis extracts alone; Group 2= P. ligulatus extracts alone Group 3=a mixture of O. guineensis + P. ligulatus extracts

Table 2. Mean diameters (mm) of zones of inhibition for the various extracts of the millipedes and the control commercial antibiotic using the agar well diffusion assay.

Groups Solvents Test bacteria MSSA ATCC 25923 MSSA ATCC 43300 1 Ethanol (99%) 15 ± 0.30 10 ± 0.03 1 Chloroform (99%) 19 ± 0.05 15 ± 0.10 1 Ethanol (70%) 25 ± 0.13 21 ± 0.05 1 Aqueous 11 ± 0.01 NA 2 Ethanol (99%) NA NA 2 Chloroform (99%) NA NA 2 Ethanol (70%) NA NA 2 Aqueous NA NA 3 Ethanol (99%) 29 ± 0.02 24 ± 0.15 3 Chloroform (99%) 20 ± 0.01 18 ± 0.00 3 Ethanol (70%) 15 ± 0.00 11 ± 0.02 3 Aqueous 10 ± 0.00 NA Ciprofloxacin (5 µg) 28 ± 0.00 26 ± 0.00

Group 1= O. guineensis extract alone; Group 2=P. ligulatus extract alone Group 3= a mixture of O. guineensis + P. ligulatus extracts MSSA,=methicillin-sensitive Staphylococcus aureus MRSA= methicillin-resistant Staphylococcus aureus NA= not applicable

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Table 3. Mean diameters (mm) of zones of inhibition for the various extracts of the millipedes and the control commercial antibiotics using the paper disc diffusion assay

Groups Solvents Test bacteria MSSA ATCC 25923 MRSA ATCC 43300 1 Ethanol (99%) NA NA 1 Chloroform (99%) 10 ± 0.13 NA 1 Ethanol (70%) NA NA 1 Aqueous NA NA 2 Ethanol (99%) NA NA 2 Chloroform (99%) NA NA 2 Ethanol (70%) NA NA 2 Aqueous NA NA 3 Ethanol (99%) 15 ± 0.32 12 ± 0.07 3 Chloroform (99%) NA NA 3 Ethanol (70%) NA NA 3 Aqueous NA NA Controls Ciprofloxacin (5 µg) 28 ± 0.10 26 ± 0.05 Gentamicin (10 µg) 26 ± 0.30 20 ± 0.00 Erythromycin (15 µg) 28 ± 0.18 12 ± 0.04 Tetracycline (30 µg) 26 ± 0.21 12 ± 0.11 Vancomycin (30 µg) 21 ± 0.05 16 ± 0.01

Group 1= O. guineensis extract alone; Group 2= P. ligulatus extract alone Group 3= a mixture of O. guineensis + P. ligulatus extracts MSSA= methicillin-sensitive Staphylococcus aureus MRSA= methicillin-resistant Staphylococcus aureus NA= not applicable

Table 4. MIC and MBC values (mg/ml) of ethanol extract of millipedes (Group 3) against the bacterial isolates

Test bacteria Group 3 extracta Control*

MIC MBC MIC MBC

MSSA 1.56 6.25 0.00019 0.391

MRSA 6.25 25.00 0.00076 1.563 aGroup 3 extract= mixture of O. guineensis + P. ligulatus extract *Ciprofloxacin MSSA= methicillin-sensitive Staphylococcus aureus MRSA= methicillin-resistant Staphylococcus aureus

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a b

Plate: 1a, b, The Chocolate-Banded millipede, Ophistreptus guineensis (Silvestri). (Photos & ID: M.K. BILLAH, 2014).

Plate: 1c, The Chocolate-Banded millipede, Ophistreptusguineensis (Silvestri). (Photos & ID: M.K. BILLAH, 2014).

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Plate 2. Pachybolus ligulatus

(Photos & ID: M.K. BILLAH, 2014).

REFERENCES [ 6 ] Hiramatsu, K., Katayama, Y. and Yuzawa, H., 2002. Molecular genetics of methicillin- [ 1 ] Anonymous, 2011. Animal Biodiversity. resistant Staphylococcus aureus. Int J Med Zootaxa 3148, 159-164. Microbiol 292, 67-74. [ 2 ] Sierwald, P. and Bond, J.E., 2007. Current [ 7 ] Zhang, K., Sparling, J., Chow, B.L., Status of the Myriapod Class Diplopoda Elsayed, S., Hussain, Z., Church, D.L., et al., (Millipedes): Taxonomic Diversity and 2004. New quadriplex PCR assay for detection Phylogeny. Annu Rev Entomol 52(1): 401-420. of methicillin and mupirocin resistant and [ 3 ] Kubo, T., Fujita, K., Nihei, K. and simultaneous discrimination of Masuoka, N., 2003. Non-antibiotic Staphylococcus aureus from coagulase- antibacterial activity of dodecyl gallate. negative staphylococci. J Clin Microbiol 42, Bioorg Med Chem 11, 573-580. 4947-4955. [ 4 ] Enright, M.C., 2003. The evolution of a [ 8 ] Bambeke, F.V., Balzi, E. and Tulkens, resistant pathogen-the case of MRSA. Curr P.M., 2000. Antibiotic efflux pumps. Biochem Opin Pharmacol 3, 474-479. Pharmacol 60, 457-470. [ 5 ] Rod, L., Hoyt, K.S. and Hayes, K., 2007. [ 9 ] Gibbons, S., Oluwatuyi, M. and Kaatz, Methicillin-resistant Staphylococcus aureus G.W., 2003. A novel inhibitor of multidrug (MRSA) Infection. Adv Emerg Nurs J 29, 118- efflux pumps in Staphylococcus aureus. J 128. Antimicrob Chem 51, 13-17.

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[ 10 ] Kumar, A. and Schweizer, H.P., 2005. Informational Supplement CLSI document Bacterial resistance to antibiotics: Active M100-S9. CLSI, Wayne, PA. efflux and reduced uptake. Adv Drug Deliv [ 17 ] Andrews, J.M., 2005. BSAC Standard Disc Rev 57, 1486-1513. Susceptibility Testing Method (version 4). J [ 11 ] Cepeda, J.A., Whitehouse, T., Cooper, B., Antimicrob Chemother 56, 60-76. Hails, J., Jones, K. and Kwaku, F., 2005. [ 18 ] Cheesbrough, M., 2006. District Isolation of patients in single rooms or Laboratory Practice in Tropical Countries cohorts to reduce spread of MRSA in Part 2. 2nded. New York: Cambridge intensive-care units: Prospective two-centred University Press. study. Lancet 365, 295-304. [ 19 ] Xylander, W.E.R., 2009. Antibacterial [ 12 ] Ghana News Agency (GNA, 2012). substances and characterization of the Management of the Korle Bu Teaching haemolymph of Chilopoda and Diplopoda Hospital has closed, copied to the Ghana (, Arthropoda). Soil Organisms News Agency (GNA) in Accra on Saturday. 81(3), 413-429. Available at: [ 20 ] Valderrama, X., Robinson, J.G., Attagalle, www.ghananewsagency.org/.../K-Bu-children- A.B. and Eisner, T., 2000. Seasonal anointing s-emergency-ward-closed/?[Accessed on with millipedes in a wild primate: A chemical 28thJanuary 2012]. defense against insects? J Chem Ecol 26(12), [ 13 ] Pesewu, G.A., Dogbe, R., Asmah, R.H., 2781-2783. Olu-Taiwo, M.A. and Adjei, D.N., 2014. [ 21 ] Zito, M., Evans, S. and Weldon, P.J., 2003. Prevalence and susceptibility profiles of Owl monkeys (Aotus spp) self-anoint with methicillin-resistant Staphylococcus aureus plants and millipedes. Folia Primatol 74, 159- (MRSA) in the University of Ghana Hospital, 161. Legon, Accra, Ghana. Int J Pharm Bio Sci [ 22 ] Weldon, P.G., 2004. Defensive anointing: 5(3), (B)185-93. Extending chemical phenotype and [ 14 ] Wang, L. and Barret, J.F., 2007. Control unorthodox ecology. Chemoecology 14, 1-4. and Prevention of MRSA infections. Methods [ 23 ] Carroll, J.F., Kramer, M., Weldon, P.J. Mol Biol 391, 209-225. and Robbins, R.G., 2005. Anointing chemicals [ 15 ] Pemberton, R.W., 1999. Insects and other and ectoparasites: Effects of benzoquinones arthropods used as drugs in Korean from millipedes on the lone star tick, traditional medicine. J Ethnopharmacol 65(3), Amblyomma americanum. J Chem Ecol 31(1), 207-216. 63-64. [ 16 ] Clinical Laboratory Standard Institute [ 24 ] Roncadori, R.W., Duffey, S.S. and Blum, (CLSI, 2007): Performance Standards for M.S., 1985. Antifungal activity of defensive Antimicrobial Susceptibility Testing: Ninth secretions of certain millipedes. Mycologia 72(2), 185-191.

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[ 25 ] Ngbolua, K.N., Ngunde-te-Ngunde, S., Tshidibi, D.J., Lengbiye, M.E., Mpiana, T.P., Ekutsu, E.G., et al., 2014. Anti-sickling and antibacterial activities of extracts from a Congolese Diplopod (Tachypodoiulus sp., Arthropoda). J Adv Bot Zool 1(3), 1-5.

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