Pharmacology & Pharmacy, 2017, 8, 306-323 http://www.scirp.org/journal/pp ISSN Online: 2157-9431 ISSN Print: 2157-9423 DOI: 10.4236/pp.2017.89023 Sep. 29, 2017 306 Pharmacology & Pharmacy In vitro Antibacterial Efficacy of Bidens pilosa, Ageratum conyzoides and Ocimum suave Extracts against HIV/AIDS Patients’ Oral Bacteria in South-Western Uganda Joseph Obiezu Chukwujekwu Ezeonwumelu1,2,3,10*, Muhammad Ntale4, Steve Okwudili Ogbonnia5, Ezera Agwu6,12, Julius Kihdze Tanayen7,3, Keneth Iceland Kasozi8, Chukwudi Onyeka Okonkwo9, Anthonia Shodunke2, Ambrose Amamchukwu Akunne2,10, Onokiojare Ephraim Dafiewhare10, Jennifer Chibuogwu Ebosie2,10, Frederick Byarugaba11 1Department of Pharmacy, Faculty of Medicine, Mbarara University of Science and Technology, Mbarara, Uganda 2Department of Clinical and Biopharmacy, School of Pharmacy, Kampala International University Western Campus, Ishaka, Uganda 3Kampala International University Complementary and Alternative Medicine Research (KIUCAMRES) Group, Ishaka, Uganda 4Department of Chemistry, Makerere University, Kampala, Uganda 5Department of Pharmacognosy, Faculty of Pharmacy, University of Lagos, Lagos, Nigeria 6Department of Microbiology, Faculty of Biomedical Sciences, Kampala International University Western Campus, Ishaka, Uganda 7Department of Pharmacology and Therapeutics, Faculty of Medicine, Mbarara University of Science and Technology, Mbarara, Uganda 8Department of Physiology, Faculty of Biomedical Sciences, Kampala International University Western Campus, Ishaka, Uganda 9Department of Human Physiology, Faculty of Basic Medical Sciences, College of Medicine, Nnewi Campus, Nnamdi Azikiwe University, Nnewi, Nigeria 10Department of Public Health, Faculty of Applied Sciences, Bishop Stuart University, Mbarara, Uganda 11Department of Microbiology, Faculty of Medicine, Mbarara University of Science and Technology, Mbarara, Uganda 12Department of Medical Microbiology and Immunology, University of Kabale, Kabale, Uganda Abstract The objective of the study was to determine the antibacterial efficacy of Bidens pilosa Aqueous (BPA), Bidens pilosa Ethanolic (BPE), Ageratum conyzoides Aqueous (ACA), Ageratum conyzoides Ethanolic (ACE), Ocimum suave Aqueous (OSA) and Ocimum suave Ethanolic (OSE) extracts on HIV/AIDS patients’ oral bacteria. Healthy green leaves of the plants were collected in Ishaka Uganda, processed and portions separately extracted with hot distilled water and cold ethanol. The susceptibility, MIC and MBC of each extract were determined using standard protocols. The bacteria had significant (p < 0.05) respective total susceptibilities of 35 [28.7%] to BPA; 42 [34.4%] to BPE; 61 How to cite this paper: Ezeonwumelu, J.O.C., Ntale, M., Ogbonnia, S.O., Agwu, E., Tanayen, J.K., Kasozi, K.I., Okonkwo, C.O., Shodunke, A., Akunne, A.A., Dafiewhare, O.E., Ebosie, J.C. and Byarugaba, F. (2017) In vitro Antibacterial Efficacy of Bidens pilosa, Ageratum conyzoides and Ocimum suave Extracts against HIV/AIDS Patients’ Oral Bacteria in South-Western Uganda. Pharmacology & Pharmacy, 8, 306-323. Copyright © 2017 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://www.scirp.org/journal/pp https://doi.org/10.4236/pp.2017.89023 http://www.scirp.org http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.4236/***.2017.***** J. O. C. Ezeonwumelu et al. DOI: 10.4236/pp.2017.89023 307 Pharmacology & Pharmacy [50.0%] to ACA; 45 [36.9%] to ACE; 38 [31.1%] to OSA; 32 [26.3%] to OSE; 105 (86.0%)] to ceftriaxone. BPE, ACA, OSA, OSE and ceftriaxone had signif- icant MIC with [F(1, 13); P = 0.00 and BPA with F(1, 13); P = 0.03]. BPE, ACA, ACE, OSA and ceftriaxone had significant MBC with [F(1, 13); P = 0.00 and BPA with F(1, 13); P = 0.01] on the test bacteria (MANOVA). These tested medicinal plants’ extracts and ceftriaxone had significant activity against oral bacteria with ACA having the best activity when compared with the control. However, the plants’ extracts were resisted by some of the bacte- ria. These findings validate the claims of efficacy of Bidens pilosa, Ageratum conyzoides and Ocimum suave on oral lesions of HIV/AIDS patients made by traditional healers and local people in South-Western Uganda. We recom- mend a detailed study of structural identities and activities of the active anti- bacterial principle(s) in these plants for possible new drug entities and verifi- cation of the interactive effects of the principle(s) with ARVs and cotrimox- azole used daily by HIV/AIDS patients. Keywords Antibacterial Efficacy, Bidens Pilosa, Ageratum Conyzoides, Ocimum Suave, Oral Bacteria, HIV/AIDS, Uganda 1. Introduction Many drugs owe their sources more to higher plants including Bidens pilosa, Ageratum conyzoides and Ocimum suave. than bacteria and fungi. Plants-derived broad spectrum antimicrobials are of high clinical value in the treatment of re- sistant microbial infections [1]. Low level of hygiene and sanitation in develop- ing countries is responsible for increased widespread bacterial and fungal infec- tions [2]. There were wide reports of death of about 300,000 children yearly from various under-developed regions of the globe due to diarrhoea commonly caused by Escherichia coli, Shigella spp., Salmonella spp., and Yersianiaspp [3] [4]. HIV/AIDS associated infections have emerged with resistant aetiology among HIV positive UTI patients in urban Nigeria [5], and among HIV positive oral lesion patients in rural and semi-urban Uganda [6] [7] [8]. To combat the public health burden, the use of local plants with self-defensive antimicrobial phytochemicals by local communities with poor socio-economic status becomes inevitable [1]. Such plants serve as attractive alternative to expensive orthodox drugs. These plants include, but not limited to, Bidens pilosa, Ageratum con- yzoides and Ocimum suave. Bidens pilosa is a member of the Asteraceae family, known for its medicinal values worldwide [9]. Bidens pilosa is called “Enyaba- rashana” in Runyankole language in Uganda and “Ogwumma” in Igbo language in Nigeria. Bidens pilosa is a cosmopolitan, annual herb which originates from tropical Africa, America and Asia, where its roots, leaves, and seeds are reported to have antimicrobial and many other medicinal values [9]. Polyacetylenes from http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.4236/pp.2017.89023 Received: May 24, 2017 Accepted: September 26, 2017 Published: September 29, 2017 Open Access https://doi.org/10.4236/pp.2017.89023 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.4236/pp.2017.89023 J. O. C. Ezeonwumelu et al. DOI: 10.4236/pp.2017.89023 308 Pharmacology & Pharmacy Bidens pilosa have antimicrobial activity while some flavonoids have an- ti-inflammatory properties [10] [11] [12]. Ageratum conyzoides is a member of Asteraceae family, native to Central America, Carribean, Florida (USA), South- east Asia, South China, India, West Africa, Australia and South America [13]. A. conyzoides is called “Butabuta” in Runyankole language in Uganda and “Eghu- eri-okukoatu” in Igbo language in Nigeria. The extracts have been reported to have very good antimicrobial activity. A. conyzoides has been used in folklore for the treatment of fever, pneumonia, cold, rheumatism, spasm, headache, wounds and burns [14] [15]. Indians use this species as a bacteriocide, an- ti-dysenteric, and antilithic [16]. In Asia, South America, and Africa, aqueous extract of this plant is used as a bacteriocide [17] [18] [19]. The antibacterial in- hibitory activities of A. conyzoides in the development of Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa are known [15] [18] [20]. Ocimum suave (in the family of Lamiacaeae) and its other numerous species are native to African region (especially Tanzania and the Zanzibar arc- hipelago, Uganda, Kenya, Ethiopia, Nigeria, Ghana, Burkina Faso, South Africa) and South Asian region [21]. It is commonly called African Bush Basil, “Omu- jaaja” in Runyankole language in Uganda and “Nchanwu” in Igbo language in Nigeria. It is widely used as an antiseptic, antibacterial and for many other pur- poses [22]-[33]. The use of Bidens pilosa, Ageratum conyzoides and Ocimum suave as local alternative drug to treat oral bacterial lesions warrants scientific validation in line with the principles of universal primary health care services to the communities. Therefore, the objective of the study was to validate the scien- tific evidence for the use of Bidens pilosa, Ageratum conyzoides and Ocimum suave in the treatment of oral lesions of HIV/AIDS patients in South-Western Uganda. 2. Materials and Methods 2.1. Drugs and Reagents These included ceftriaxone susceptibility discs (Biomerieux® France) and ethanol (Lobachemie® South Africa). 2.2. Study Design The study was experimental in design. Clinical oral bacterial isolates [6] from the Microbial Bank, Department of Microbiology and Immunology, Kampala International University-Western Campus (KIU-WC) were supplied by the Unit for analysis. 2.3. Sample Size and Sampling Technique Sample size of 100 out of the available 610 clinical bacterial isolates [6] were se- lected using systematic-random sampling technique in a 1:6 ratio. Subsequently, a duplicate of each of the selected isolates was made to serve as a backup. Then https://doi.org/10.4236/pp.2017.89023 J. O. C. Ezeonwumelu et al. DOI: 10.4236/pp.2017.89023 309 Pharmacology & Pharmacy 22 isolates of the standard bacteria (a pair) were selected to serve as control for the 11 species of bacterial isolates under study based on whether the selected isolate was a gram negative or gram positive bacterium. This made a grand total of 122 bacterial isolates [7]. 2.4. Confirmation of Bacterial Viability and Identity MacConkey agar, Chocolate agar and Blood agar were used as the primary me- dia to grow the 122 samples to get fresh viable isolates. The viability of the pre- viously identified isolates and standard bacteria were confirmed by growing the isolates on MacConkey agar, Chocolate agar and Blood agar [34] [35] [36] [37] [38]. To further confirm microbial identities, Chrom-agar orientation and car- bohydrate assimilation tests were done using the analytical profile index (API) testing kits (Biomerieux® SA France, INS005517) and results were read using apiweb TM identification software. 2.5. Selection and Identification of Plants for Antibacterial Activity Testing A survey was conducted in Ishaka and its environs which showed that 97.0 (80.8%), 118.0 (98.3%) and 95.0 (79.2%) out of 120 traditional healers and local people respectively used Bidens pilosa, Ageratum conyzoidesand Ocimum suave to treat oral lesions associated with HIV/AIDS disease [unpublished data]. A sample of each of the plants was locally identified by the traditional healers and local users of the herbs. The plants were botanically identified by a botanist at the Department of Biology of Mbarara University of Science and Technology, Mbarara, Uganda and their voucher specimens numbered MUST-DB-0001, MUST-DB-0002 and MUST-DB-0003 respectively for Bidens pilosa, Ageratum conyzoides and Ocimum suave were deposited in the Departmental Herbarium for reference purposes. 2.6. Collection, Processing and Preparation of Plant Extract Fresh healthy, green leaves of Bidens pilosa, Ageratum conyzoides and Ocimum suave were collected from Ishaka area of Western Uganda during rainy season in February 2015. The method of extraction used by traditional healers and local users of the herbs were replicated in this study. The aqueous extract was made with 200 g of the powdered plant material in 1 Litre of distilled water for an hour hot extraction. The ethanolic extract was made with 200 g of the plant material in 1Litre of absolute ethanol for a 72-hour cold extraction. The filtrate of the aqueous extract was dried in a water bath at 100˚C whereas the ethanolic extract filtrate was dried in a hot air oven at 40˚C. The dried extracts were then stored at −20˚C without exposing them to freeze-thaw cycles and humidity [39]. The aqueous extract was always freshly reconstituted in distilled water while ethanol- ic extract suspension was always reconstituted with minute quantity of Tween 80 in distilled water for use in the studies. https://doi.org/10.4236/pp.2017.89023 J. O. C. Ezeonwumelu et al. DOI: 10.4236/pp.2017.89023 310 Pharmacology & Pharmacy 2.7. Susceptibility Testing An overnight culture was used to make a bacterial suspension corresponding to a 1.5 McFarland standard whose turbidity was estimated with a densitometer for precision. The suspension was then homogenized and used immediately to de- termine the sensitivity, MIC and MBC of all the purified isolates according to the methods defined by Cheesbrough [34]. The diluted extracts and ceftriaxone discs were used to determine their antibacterial activities (bacterial sensitivity and resistance patterns on isolates) in accordance with the CLSI modified Kir- by-Buaer tube dilution and agar well diffusion methods [40]. The standard ref- erence organisms of the American Type Culture Collection such as ATCC 25923 Staphylococcus aureus, ATCC 25922 Escherichia coli and ATCC 27853 Pseu- domonas aeruginosa were selected as the controls for pyogenic bacteria, entero- bacteriaceae and Pseudomonas aeruginosa respectively. 2.8. Broth Preparation for MIC Mueller Hinton agar (MHA) was prepared and tested for sterility overnight. Freshly prepared suspension made from 18 - 24 hour sub-cultured bacterial strains was inoculated on the MHA. Sterile cork borer was used to make holes on the MHA. Then, the sensitivity and resistance patterns, MIC and MBC were determined following the standard conventional method [34]. The specific con- centrations of a stock solution of the extracts “BPA, BPE, ACA, ACE, OSA and OSE” (0.5 g/mL); and ceftriaxone injection (200 mg/mL) were prepared by di- luting in 1mL of water for injection in sterile plain tubes. Then, a 200 µL of the stock solution of the extracts and ceftriaxone was each pipetted separately into each of the holes made on the media, incubated for 18 - 24 hours and then zones of inhibition were determined using a transparent ruler and recorded for deter- mination of sensitivity and resistance patterns. Afterwards, serial dilutions ranging from 0.5 to 0.004 strength of each of the stock solutions were prepared with 1mL of distilled water in each case [41] [42]. Then, micropipettes were used to add 200 µL of each dilution into respective holes on the agar plate for agar well diffusion beginning with the lowest dilution. The plates and the tubes were incubated at 37˚C for 18 - 24 hours, checked for growth and zone of inhibition in the petri dishes and then recorded. 2.9. MIC and MBC Determination MIC was determined by recording the smallest concentration of the drug that inhibited growth of microorganisms. The prevalent high turbidity in the tubes did not permit direct determination of MBC. Samples were therefore taken with sterile swab sticks from cleared zones of inhibition considering each specific di- lution on the petri dishes and then smeared onto a fresh MHA medium and in- cubated for 18 - 24 hours and then results were checked for MBC as the smallest concentration of the drug that killed the organisms which corresponds with the plate in which the smallest concentration of the drug did not permit the re- growth of the organisms [41]. https://doi.org/10.4236/pp.2017.89023 J. O. C. Ezeonwumelu et al. DOI: 10.4236/pp.2017.89023 311 Pharmacology & Pharmacy 2.10. Data Analysis The MS Excel 2010 version was used to enter the duplicates of the data and transferred to SPSS Version 20.0 for analysis. Results were expressed as mean ± SD. Chi square test was used to test the significance (P < 0.05) of susceptibility patterns of the oral bacteria to the medicinal plants’ extracts and ceftriaxone. MANOVA, test of between subject effects and pairwise comparisons were done to determine variations among groups in MIC and MBC tests with P < 0.05 con- sidered as significant. 3. Results 3.1. Susceptibility of Oral Bacteria In susceptibility testing of 44 isolates of Staphylococcus aureus (Table 1), Bidens pilosa, Ageratum conyzoides and Ocimum suave extracts were found to be ac- tive on Staphylococcus aureus. More importantly, 24 [54.5%] of isolates of Sta- phylococcus aureus were susceptible to aqueous extract of Ageratum conyzoides. Thirty three [75%] of the Staphylococcus aureus isolates were fully vulnerable to the effect of ceftriaxone which was a reference antibiotic. Staphylococcus sapro- phyticus was 100% susceptible to aqueous extract of Bidens pilosa, aqueous ex- tract of Ageratum conyzoides, ethanolic extract of Ageratum conyzoides and ceftriaxone. Streptococcus mutans isolates were found to be reasonably sensitive to aqueous extract of Ageratum conyzoidesat 5 [55.6%]; to ethanolic extract of Ocimum suaveat 6 [66.7%] and to ceftriaxone at [8 (88.9%)]. Streptococcus pneumoniae had full susceptibility of 21 [100%] to ceftriaxone and majorly of 5 [23.8%] susceptibility to ethanolic extract of Ageratum conyzoides. The isolate of non-haemolytic streptococcus showed full susceptibility [100%] to aqueous and ethanolic extracts of Bidens pilosa and ceftriaxone. Bacillus cereus was to- tally sensitive [100%] to aqueous extract of Ageratum conyzoides, ethanolic Ocimum suave and ceftriaxone. All the isolates of Staphylococcus aureus ATCC 25293 showed 100% susceptibility to aqueous and ethanolic Bidens pilosa, Age- ratum conyzoides and Ocimum suave. Five [55.6%] isolates of E. coli were sus- ceptible to ethanolic extract of Bidens pilosa. Three [33.3%] isolates of E. coli were sensitive to ethanolic extract of Ageratum conyzoides. E. coli was found to be 6 [66.6%] susceptible to ceftriaxone. Salmonella pullorum was only sensitive to ethanolic extract of Bidens pilosa. Aqueous extract of Ageratum conyzoides and ceftriaxone respectively exhibited major activities on Klebsiella pneumoniae at 7 [77.8%] and 8 [88.9%]. Proteus mirabilis were 100% susceptible to ceftriax- one and 50% susceptible to ethanolic extract of Ageratum conyzoides. The ref- erence E. coli ATCC 25922 had 8 [100%] susceptibility to all the extracts and ceftriaxone. Pseudomonas aeruginosa were 100% susceptible to ceftriaxone; and 50% sensitive to ethanolic extracts of Ageratum conyzoides and Ocimum suave. Conversely, Pseudomonas aeruginosa ATCC 27853 demonstrated 100% suscep- tibility to all the medicinal plants in addition to ceftriaxone. https://doi.org/10.4236/pp.2017.89023 J. O. C. Ezeonwumelu et al. DOI: 10.4236/pp.2017.89023 312 Pharmacology & Pharmacy Table 1. Susceptibility patterns of oral bacterial isolates to antibacterial medicinal plants’ extract/agent. Bacterium No. of isolates S/R/I Antibacterial medicinal plants’extract/agent (No (%)) BPA (0.5 g) BPE (0.5 g) ACA (0.5 g) ACE (0.5 g) OSA (0.5 g) OSE (0.5 g) Ceftri (30 µg) Staphylococcus aureus 44 S 7 (15.9) 9 (20.5) 24 (54.5) 10 (22.7) 9 (20.5) 1 (2.3) 33 (75.0) R 36 (81.8) 35 (79.5) 0 (0.0) 22 (50.0) 34 (77.2) 33 (75.0) 11 (25.0) I 1 (2.3) 0 (0.0) 20 (45.5) 12 (27.3) 1 (2.3) 10 (22.7) 0 (0.0) Staphylococcus aureus ATCC 25293 12 S 12 (100.0) 12 (100.0) 12 (100.0) 12 (100. 0) 12 (100.0) 12 (100.0) 11 (91.7) R − − − − − − 1 (8.3) Staphylococcus saprophyti- cus 1 S 1 (100.0) 0 (0.0) 1 (100.0) 1 (100.0) 0 (0.0) 0 (0.0) 1 (100.0) R 0 (0.0) 1 (100.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) I 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (100.0) 1 (100.0) 0 (0.0) Escherichia coli 9 S 1 (11.1) 5 (55.6) 0 (0.0) 3 (33.3) 1 (11.1) 1 (11.1) 6 (66.7) R 8 (88.9) 3 (33.3) 0 (0.0) 0 (0.0) 4 (44.4) 5 (55.6) 0 (0.0) I 0 (0.0) 1 (11.1) 9 (100.0) 6 (66.7) 4 (44.4) 3 (33.3) 3 (33.3) Escherichia coli ATCC 25922 8 S 8 (100.0) 8 (100.0) 8 (100.0) 8 (100.0) 8 (100.0) 8 (100.0) 8 (100.0) Salmonella pullorum 1 S 0 (0.0) 1 (100.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) R 1 (100.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (100.0) 1 (100.0) 1 (100.0) I 0 (0.0) 0 (0.0) 1 (100.0) 1 (100.0) 0 (0.0) 0 (0.0) 0 (0.0) Klebsiella pneumoniae 9 S 0 (0.0) 2 (22.2) 7 (77.8) 1 (11.1) 0 (0.0) 0 (0.0) 8 (88.9) R 9 (100.0) 7 (77.8) 0 (0.0) 7 (77.8) 8 (88.9) 7 (77.8) 0 (0.0) I 0 (0.0) 0 (0.0) 2 (22.2) 1 (11.1) 1 (11.1) 2 (22.2) 1 (11.1) Streptococcus mutans 9 S 1 (11.1) 2 (22.2) 5 (55.6) 1 (11.1) 4 (44.4) 6 (66.7) 8 (88.9) R 8 (88.9) 7 (77.8) 1 (11.1) 8 (88.9) 5 (55.6) 3 (33.3) 1 (11.1) I 0 (0.0) 0 (0.0) 3 (33.3) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Streptococcus pneumoniae 21 S 2 (9.6) 0 (0.0 1 (4.8) 5 (23.8) 2 (9.6) 1 (4.8) 21 (100.0) R 19 (90.4) 18 (85.6) 7 (33.3) 16 (76.2) 18 (85.6) 19 (90.4) 0 (0.0) I 0 (0.0) 3 (14.4) 13 (61.9) 0 (0.0) 1 (4.8) 1 (4.8) 0 (0.0) Non-haemolytic streptococcus 1 S 1 (100.0) 1 (100.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (100.0) R 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (100.0) 1 (100.0) 0 (0.0) I 0 (0.0) 0 (0.0) 1 (100.0) 1 (100.0) 0 (0.0) 0 (0.0) 0 (0.0) Proteus mirabilis 2 S 0 (0.0) 0 (0.0) 0 (0.0) 1 (50.0) 0 (0.0) 0 (0.0) 2 (100.0) R 2 (100.0) 2 (100.0) 0 (0.0) 0 (0.0) 2 (100.0) 2 (100.0) 0 (0.0) I 0 (0.0) 0 (0.0) 2 (100.0) 1 (50.0) 0 (0.0) 0 (0.0) 0 (0.0) Pseudomonas aeruginosa 2 S 0 (0.0) 0 (0.0) 0 (0.0) 1 (50.0) 0 (0.0) 1 (50.0) 2 (100.0) R 2 (100.0) 1 (50.0) 2 (100.0) 0 (0.0) 2 (100.0) 1 (50.0) 0 (0.0) I 0 (0.0) 1 (50.0) 0 (0.0) 1 (50.0) 0 (0.0) 0 (0.0) 0 (0.0) Pseudomonas aeruginosa ATCC 27853 2 S 2 (100.0) 2 (100.0) 2 (100.0) 2 (100.0) 2 (100.0) 2 (100.0) 2 (100.0) Bacillus cereus Total 1 122 S 0 (0.0) 0 (0.0) 1 (100.0) 0 (0.0) 0 (0.0) 1 (100.0) 1 (100.0) R 0 (0.0) 1 (100.0) 0 (0.0) 0 (0.0) 1 (100.0) 0 (0.0) 0 (0.0) I S R I 1 (100.0) 35 (28.7) 85 (69.7) 2 (1.6) 0 (0.0) 42 (34.4) 75 (61.5) 5 (4.1) 0 (0.0) 61 (50.0) 8 (6.6) 51 (41.8) 1 (100.0) 45 (36.9) 53 (43.4) 24 (19.7) 0 (0.0) 38 (31.1) 76 (62.3) 8 (6.6) 0 (0.0) 32 (26.3) 73 (59.8) 17 (13.9) 0 (0.0) 105 (86.0) 13 (10.7) 4 (3.3) KEY: BPA = Bidens pilosa Aqueous, BPE = Bidens pilosa Ethanol, ACA = Ageratum conyzoides Aqueous, ACE = Ageratum conyzoides Ethanol, OSA = Ocimum suave Aqueous and OSE = Ocimum suave Ethanol. https://doi.org/10.4236/pp.2017.89023 J. O. C. Ezeonwumelu et al. DOI: 10.4236/pp.2017.89023 313 Pharmacology & Pharmacy In summary, all the bacteria used for the test were susceptible; 61 [50%] to aqueous extract of Ageratum conyzoides, 45 [36.9%] to ethanolic extract of Ageratum conyzoides, 42 [34.4%] to ethanolic extract of Bidens pilosa, 38 [31.1%] to aqueous extract of Ocimum suave. 35 [28.7%] to aqueous extract of Bidens pilosa, 32 [26.3%] to Ocimum suave and 105 [86.0%] to ceftriaxone (Table 1). The Pearson Chi-square test with [P = 0.00]; Likelihood ratio test with [P = 0.00] for medicinal plants; and [P = 0.03] for antibacterial agent respectively revealed significant antibacterial effects for all the medicinal plants and cef- triaxone. 3.2. Minimum Inhibitory Concentration (MIC) From Table 2, the results of the tests revealed that Streptococcus mutans, Strep- tococcus pneumoniae, Escherichia coli, Staphylococcus aureus, Staphylococcus saprophyticus, Bacillus cereus and non-haemolytic streptococcus were sensitive to aqueous extract of Bidens pilosa with a mean range of MICs from 0.01 ± 0.04 µg/ml to 1.00 ± 0.00 µg/ml. Streptococcus mutans, Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Salmonella pullorum and Klebsiella pneumoniae were sensitive to the ethanolic extract of Bidens pilosa with a mean range of MICs from 0.04 ± 0.0 µg/ml and 1.00 ± 0.00 µg/ml. The antibacterial activities of the ethanolic extract of Bidens pilosa were generally better than those of the aqueous extract of Bidens pilosa. Aqueous extract of Ageratum con- yzoides minimally and sensitively inhibited the growth of Salmonella pullorum, Klebsiella pneumoniae, Pseudomonas aeruginosa and Bacillus cereusat 0.06 ± 0.00 µg/ml. The ethanolic extract of Ageratum conyzoides minimally and sensi- tively inhibited the growth of Streptococcus mutans at 0.02 ± 0.03 µg/ml), Kleb- siella pneumoniae at 0.13 ± 0.00 µg/ml, Salmonella pullorum at 0.13 ± 0.00 µg/ml and Proteus mirabilis at 0.13 ± 0.00 µg/ml. The aqueous extract of Oci- mum suavehada minimal sensitive inhibition of growth of Staphylococcus au- reus at 0.07 ± 0.1 6 µg/ml, Streptococcus mutans at 0.17 ± 0.18 µg/ml and Strep- tococcus pneumoniae at 0.06 ± 0.13 µg/ml. The ethanolic extract of Ocimum suave had MICs against Staphylococcus saprophyticus at 0.06 ± 0.16 µg/ml, Streptococcus pneumoniae at 0.06 ± 0.15 µg/ml, E. coli at 0.50 ± 0.35 µg/ml and Klebsiella pneumoniae at 0.06 ± 0.17 µg/ml. Ceftriaxone was observed to have better MICs against most of the tested bacteria than the extracts. This implies that all the aqueous and ethanolic extracts of Bidens pilosa, Ageratum con- yzoides, and Ocimum suave minimally inhibited the growth of most oral bacte- ria (Table 2). Multiple analysis of variance of the MICs using any of the Pillai’s Trace, Wilk’s Lambda, Hotelling’s Trace or Roy’s Largest Root tests showed a high significant activity against the bacteria tested [P = 0.000], with an observed power of 100%, thus we reject the null which says there is no observed antibac- terial activity of antibacterial agents against the bacteria tested; and accept the alternative hypothesis which says that antibacterial agents have antibacterial ac- tivity against the tested bacteria. Multivariate effects of plant extracts against https://doi.org/10.4236/pp.2017.89023 J. O. C. Ezeonwumelu et al. DOI: 10.4236/pp.2017.89023 314 Pharmacology & Pharmacy Table 2. MIC of local antibacterial medicinal plants and commercial agent against oral bacterial isolates. Bacterium Minimum inhibitory concentration of locally used medicinal plants (Mean ± SD (µg/ml)) BPA BPE ACA ACE OSA OSE CEFTRI WATER/ETH Staphylococcus aureus Staphylococcus aureus ATCC 25293 Staphylococcus saprophyticus 0.16 ± 0.37 0.31 ± 0.20 0.50 ± 0.00 0.08 ± 0.18 0.13 ± 0.00 0.50 ± 0.00 0.24 ± 0.17 0.06 ± 0.00 0.13 ± 0.00 0.15 ± 0.20 0.06 ± 0.00 0.25 ± 0.00 0.07 ± 0.16 0.25 ± 0.00 0.50 ± 0.00 0.00 ± 0.00 0.16 ± 0.10 0.00 ± 0.00 0.13 ± 0.00 0.06 ± 0.00 0.00 ± 0.00 0.06 ± 0.16 0.06 ± 0.00 0.00 ± 0.00 Escherichia coli 0.12 ± 0.33 0.08 ± 0.08 0.10 ± 0.15 0.26 ± 0.20 0.18 ± 0.24 0.50 ± 0.35 0.11 ± 0.08 0.00 ± 0.00 Escherichia coli ATCC 25922 0.10 ± 0.00 0.10 ± 0.00 0.06 ± 0.00 0.13 ± 0.00 0.13 ± 0.00 0.50 ± 0.00 0.06 ± 0.00 0.00 ± 0.00 Salmonella pullorum 0.00 ± 0.00 0.10 ± 0.00 0.06 ± 0.00 0.13 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.13 ± 0.00 0.00 ± 0.00 Klebsiella pneumoniae 0.00 ± 0.00 0.08 ± 0.16 0.06 ± 0.00 0.06 ± 0.09 0.00 ± 0.00 0.06 ± 0.17 0.11 ± 0.08 0.00 ± 0.00 Streptococcus mutans 0.01 ± 0.04 0.04 ± 0.06 0.11 ± 0.15 0.02 ± 0.03 0.17 ± 0.18 0.18 ± 0.19 0.04 ± 0.03 0.00 ± 0.00 Streptococcus pneumoniae 0.06 ± 0.22 0.11 ± 0.20 0.11 ± 0.17 0.09 ± 0.16 0.06 ± 0.13 0.06 ± 0.15 0.07 ± 0.04 0.00 ± 0.00 Non-haemolytic streptococcus 1.00 ± 0.00 1.00 ± 0.00 0.50 ± 0.00 0.13 ± 0.00 0.50 ± 0.00 0.25 ± 0.00 0.13 ± 0.00 0.00 ± 0.00 Proteus mirabilis 0.00 ± 0.00 0.00 ± 0.00 0.25 ± 0.00 0.13 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.06 ± 0.00 0.00 ± 0.00 Pseudomonas aeruginosa 0.00 ± 0.00 0.25 ± 0.35 0.06 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.25 ± 0.35 0.03 ± 0.00 0.00 ± 0.00 Pseudomonas aeruginosa ATCC 27853 0.13 ± 0.00 0.13 ± 0.00 0.06 ± 0.00 0.06 ± 0.00 0.06 ± 0.00 0.06 ± 0.00 0.03 ± 0.00 0.00 ± 0.00 Bacillus cereus 0.50 ± 0.00 0.00 ± 0.00 0.06 ± 0.00 0.13 ± 0.00 0.50 ± 0.00 0.13 ± 0.00 0.06 ± 0.00 0.00 ± 0.00 KEY: BPA=Bidens pilosa Aqueous, BPE = Bidens pilosa Ethanol, ACA = Ageratum conyzoides Aqueous, ACE = Ageratum conyzoides Ethanol, OSA = Ocimum suave Aqueous and OSE = Ocimum suave Ethanol, CEFTRI = Ceftriaxone; WATER = Distilled Water and ETH = Ethanol. bacteria dependent on linearly independent pairwise comparisons among the es- timated marginal means were done. From the tests results of in-between subject effects and pairwise comparison tests when P < 0.05 was rated as statistically sig- nificant, it showed that BPE. ACA, OSA, OSE and ceftriaxone with F statistic [1, 13]; P = 0.00 and BPA with F statistic [1, 13]; P = 0.03 had significant bacterial inhibitory effects compared to ACE with F statistic [1, 13]; P = 0.24 without sig- nificant bacterial inhibitory effects. 3.3. Minimum Bactericidal Concentration (MBC) From Table 3, the best mean bactericidal activity of aqueous extract of Bidens pilosa against Streptococcus pneumoniae was 0.05 ± 0.22 µg/ml, against Staphy- lococcus aureus was 0.16 ± 0.37 µg/ml and against E. coli was 1.00 ± 0.00 µg/ml. At higher doses, it also had antibactericidal activity on Staphylococcus sapro- phyticus, Salmonella pullorum, non-haemolytic streptococcus and Pseudomonas aeruginosa. The ethanolic extract of Bidens pilosa exerted bactericidal effect on all the bacterial isolates ranging between 0.11 ± 0.32 µg/ml and 0.50 ± 0.71 µg/ml except on those of Streptococcus mutans, Proteus mirabilis and Bacillus cereus. The aqueous extract of Bidens pilosa generally had better bactericidal activity on quite a number of the bacteria used in the study than the ethanolic extract of Bi- dens pilosa. The aqueous extract of Ageratum conyzoides recorded bactericidal activity on all the bacterial isolates tested with better activity on gram positive https://doi.org/10.4236/pp.2017.89023 J. O. C. Ezeonwumelu et al. DOI: 10.4236/pp.2017.89023 315 Pharmacology & Pharmacy Table 3. MBC of local antibacterial medicinal plants and conventional agent against oral bacterial isolates. Bacterium Minimum bactericidal concentration of locally used medicinal plants Mean ± SD (µg/ml) BPA BPE ACA ACE OSA OSE CEFTRI WATER/ETH Staphylococcus aureus Staphylococcus aureus ATCC 25293 Staphylococcus saprophyticus 0.16 ± 0.37 0.06 ± 0.00 1.00 ± 0.00 0.11 ± 0.32 0.13 ± 0.00 1.00 ± 0.00 0.69 ± 0.38 0.06 ± 0.00 1.00 ± 0.00 0.50 ± 0.51 0.03 ± 0.00 0.50 ± 0.00 0.07 ± 0.16 0.06 ± 0.00 0.50 ± 0.00 0.36 ± 0.49 0.06 ± 0.0 0.00 ± 0.00 0.18 ± 0.13 0.06 ± 0.00 0.06 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 Escherichia coli 0.11 ± 0.33 0.44 ± 0.53 0.78 ± 0.44 0.89 ± 0.33 0.33 ± 0.50 0.33 ± 0.50 0.44 ± 0.24 0.00 ± 0.00 Escherichia coli ATCC 25922 0.10 ± 0.00 0.50 ± 0.00 0.13 ± 0.00 0.50 ± 0.00 0.13 ± 0.00 0.00 ± 0.00 0.03 ± 0.00 0.00 ± 0.00 Salmonella pullorum 1.00 ± 0.00 1.00 ± 0.00 1.00 ± 0.00 1.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.06 ± 0.00 0.00 ± 0.00 Klebsiella pneumoniae 0.00 ± 0.00 0.22 ± 0.44 0.69 ± 0.37 0.22 ± 0.44 0.00 ± 0.00 0.11 ± 0.33 0.44 ± 0.24 0.00 ± 0.00 Streptococcus mutans 0.00 ± 0.00 0.00 ± 0.00 0.92 ± 0.25 0.11 ± 0.33 0.17 ± 0.18 0.44 ± 0.53 0.46 ± 0.25 0.00 ± 0.00 Streptococcus pneumoniae 0.05 ± 0.22 0.14 ± 0.36 0.36 ± 0.42 0.19 ± 0.33 0.06 ± 0.13 0.14 ± 0.36 0.26 ± 0.23 0.00 ± 0.00 Non haemolytic streptococcus 1.00 ± 0.00 1.00 ± 0.00 2.00 ± 0.00 1.00 ± 0.00 0.50 ± 0.00 1.00 ± 0.00 0.25 ± 0.00 0.00 ± 0.00 Proteus mirabilis 0.00 ± 0.00 0.00 ± 0.00 1.00 ± 0.00 0.50 ± 0.71 0.00 ± 0.00 0.00 ± 0.00 0.19 ± 0.09 0.00 ± 0.00 Pseudomonas aeruginosa 0.50 ± 0.71 0.50 ± 0.71 0.63 ± 0.53 0.50 ± 0.71 0.00 ± 0.00 0.50 ± 0.71 0.31 ± 0.27 0.00 ± 0.00 Pseudomonas aeruginosa ATCC 27853 0.13 ± 0.00 0.50 ± 0.00 0.13 ± 0.00 0.13 ± 0.00 0.06 ± 0.00 0.13 ± 0.00 0.06 ± 0.00 0.00 ± 0.00 Bacillus cereus 0.00 ± 0.00 0.00 ± 0.00 0.13 ± 0.00 0.00 ± 0.00 0.13 ± 0.00 0.00 ± 0.00 0.06 ± 0.00 0.00 ± 0.00 KEY: BPA=Bidens pilosa Aqueous, BPE = Bidens pilosa Ethanol, ACA = Ageratum conyzoides Aqueous, ACE = Ageratum conyzoides Ethanol, OSA = Ocimum suave Aqueous and OSE = Ocimum suave Ethanol, CEFTRI = Ceftriaxone; WATER = Distilled Water and ETH = Ethanol. bacteria. The optimal bactericidal activity achieved by ethanolic extract of Age- ratum conyzoides against Streptococcus mutans, Streptococcus pneumoniae, Klebsiella pneumoniae, Staphylococcus saprophyticus, Staphylococcus aureus, Proteus mirabilis and Pseudomonas aeruginosa ranged from 0.11 ± 0.33 µg/ml to 0.50 ± 0.71 µg/ml. The aqueous extract of Ocimum suave killed the isolates of Streptococcus pneumoniae, Staphylococcus aureus, Bacillus cereus, Streptococ- cus mutans, E. coli, non-haemolytic streptococcus and Staphylococcus sapro- phyticus at minimum concentrations ranging from 0.06 ± 0.13 µg/ml to 0.50 ± 0.00 µg/ml. Generally, the aqueous extract of Ocimum suave exhibited better bactericidal activity on the gram positive bacteria than on the gram negative bacteria. The ethanolic extract of Ocimum suave was able to kill Klebsiella pneumoniae and Streptococcus pneumoniae at minimum concentrations of 0.11 ± 0.33 µg/ml and 0.14 ± 0.36 µg/ml. The bactericidal activity of aqueous extract of Ocimum suave was generally better than that of ethanolic extract of Ocimum suave. The aqueous extract of Ocimum suave was seen to have a relatively over- all superior bactericidal activity to aqueous extract of Bidens pilosa. ethanolic extract of Ageratum conyzoides, aqueous extract of Ageratum conyzoides, etha- nolic extract of Bidens pilosa and Ocimum suave. Ceftriaxone had better bacte- ricidal activity on Staphylococcus saprophyticus, Salmonella pullorum, Bacillus cereus, Proteus mirabilis and non-haemolytic streptococcus than the aqueous and ethanolic extracts of Bidens pilosa, Ageratum conyzoides and Ocimum https://doi.org/10.4236/pp.2017.89023 J. O. C. Ezeonwumelu et al. DOI: 10.4236/pp.2017.89023 316 Pharmacology & Pharmacy suave when compared with control organisms. Multivariate analysis using any of Pillai’s Trace, Wilk’s Lambda, Hotelling’s Trace or Roy’s Largest Root tests showed a high statistically significant bactericidal activity against the bacteria tested [P = 0.000], with an observed power of 100%, thus we reject the null hy- pothesis which says there is no observed bactericidal activity by phy- to-antibacterial agents against the bacteria tested; and hence we accept the alter- native hypothesis which says that antibacterial agents have bactericidal activity against the tested bacteria as represented in Table 3. Multivariate effects of bac- teria dependent on linearly independent pairwise comparisons among the esti- mated marginal means were done. From the results of in-between tests, it showed that BPE, ACA, ACE, OSA and ceftriaxone with F [1, 13]; P = 0.00 and BPA with F [1, 13]; P = 0.01 had significant bactericidal effects compared to OSE with F [1, 13]; P = 1.25 which did not exert any significant bactericidal effects on the test bacteria. From pairwise comparisons tests, it showed that BPA, BPE, ACA, ACE and OSA did not have statistically significant difference in bacteri- cidal activity against Salmonella pullorum, Staphylococcus saprophyticus, Bacil- lus cereus, Escherichia coli, Klebsiella pneumoniae, non-haemolytic streptococ- cus, Proteus mirabilis, Streptococcus mutans. Streptococcus pneumoniae and Staphylococcus aureus. 4. Discussion Higher plants have contributed immensely to sources of many pharmaceuticals dispensed today in pharmacies, with very limited number envisioned for anti- microbial uses because majority of antibiotics in the global market today ema- nated from bacterial and fungal sources [1]. But today, we argue that clinical microbiologists have two reasons to be interested in the antimicrobial plant ex- tracts because they inhibit microorganisms via multiple targets antithetical to antibiotics with effective short life span due to resistance from frequent and in- discriminate use of antibiotics resulting from higher rate of infections due to low level of hygiene and sanitation in Africa and so higher plant antimicrobials can be of great value in treating multi-resistant microorganisms [2]. This neglect of research on higher plant-derived antimicrobials has resulted in widespread mul- ti-resistant infections; that about 300,000 children die annually in some places owing to common intestinal diarrhoeal diseases caused by E. coli, Shigella spp., Salmonella spp., and Yersiania spp [3] [4]. In most of these affected areas, people resort to the use of local and indigenous plants as the possible option to treat the infections due to high cost and non-availability of effective conventional drugs and microbial resistance. The plants possess unlimited potentials in synthesizing aromatic secondary metabolites such as phenolics, terpenoids and essential oils, alkaloids, lectines and polypeptides, and polycetyles to defend the plants against microorganisms; amongst which phenols are the most reportedly documented ones with potential antimicrobial activity [1] [43]. Instances of these phenolic compounds include catechol or caffeic acid, quinones, flavonoids, flavonols, fla- https://doi.org/10.4236/pp.2017.89023 J. O. C. Ezeonwumelu et al. DOI: 10.4236/pp.2017.89023 317 Pharmacology & Pharmacy vones and coumarins. Tannins are examples of polymeric phenols [44]. Polyme- rization via air oxidation perhaps, is the reason for the phenolics’ observed an- timicrobial activity of the plants through iron deprivation of hydrogen bonding with vital proteins especially microbial enzymes [43]. Therefore, an important feature central to the plant phytochemicals’ toxification and detoxification is their polymerization size [45]. Many assays including agar disc diffusion or agar well diffusion as modified [46] have frequently been employed in microbiology laboratories in screening for the antimicrobial activities of plants’ extracts and serial broth microdilution assay [40] has been in use in quantifying the antimi- crobial activity of plants’ extracts and determining MIC and MBC. Many plants which have been screened in laboratories for antimicrobial activities are often selected sequel to their traditional uses which was also the case in our study. The plants were ascertained to be used in the treatment of oral lesions of HIV/AIDS patients in South-Western Uganda by traditional healers and local people. The most frequently used plants as ascertained were eventually selected for the study and they included Bidens pilosa, Ageratum conyzoides and Ocimum suave. The plant extracts were also prepared in the same way the traditional healers and lo- cals prepare them for use. Hence, the antimicrobial susceptibility and smallest concentration of each of the antibacterial medicinal plants that inhibited (MIC) or killed (MBC) the oral bacterial isolates of HIV/AIDS patients were deter- mined in the study using the agar well diffusion and serial dilution test methods as described in the study methods. The results of the study proved that higher plants can produce effective antimicrobial agents because the tested BPA, BPE, ACA, ACE, OSA and OSE extracts have antibacterial properties as claimed by the traditional healers and local people who treat oral lesions of HIV/AIDS pa- tients in South-Western Uganda with the extracts of the plants. These findings corroborate the reports that polyacetylenes from Bidens pilosa possess antimi- crobial activity while some flavonoids from the plant possess anti-inflammatory properties [9] [10] [11] [12]. The findings in this study also attest to the pre- viously reported studies on antibacterial activities of Ageratum conyzoides [20]. Another study verified its inhibitory activities against in vitro development of Staphylococcus aureus using ether and chloroform extracts [15] and this was corroborated by another study which confirmed its inhibitory activities against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas ae- ruginosa using methanolic extract of the whole plant [18]. The findings equally confirm the reports that Ocimum suave has been used in the treatment of dy- sentery, dyspepsia, skin diseases, bacterial, and fungal infections [22] [33]. The findings of the study therefore lay credence and validation to the claims made by the traditional healers and local people of South-Western Uganda that Bidens pilosa Aqueous, Bidens pilosa Ethanolic, Ageratum conyzoides Aqueous, Age- ratum conyzoides Ethanolic, Ocimum suave Aqueous and Ocimum suave Etha- nolic can be used to treat some bacteria infected oral lesions of HIV/AIDS pa- tients. The gram positive bacteria were reported often to be more susceptible to https://doi.org/10.4236/pp.2017.89023 J. O. C. Ezeonwumelu et al. DOI: 10.4236/pp.2017.89023 318 Pharmacology & Pharmacy plant extracts than the gram-negative ones [2] [47] [48]. This is in agreement with the results of our study because most of the plant extracts exhibited much better antibacterial activity on gram positive bacteria than on gram negative bacteria and Pseudomonas aeruginosa. Indeed, this greater activity on gram pos- itive bacteria might be because gram positive bacteria have only an outer pepti- doglycan layer which is not an effective barrier [49]. The gram negative bacteria have an outer phospholipid membrane that makes the cell wall impermeable to lipophilic solutes, while the porines constitute a selective barrier to hydrophilic solutes with an exclusion limit of about 600 Da [50]. The screening of extracts for antimicrobial activity in vitro often involves reference and clinical strains of microorganisms isolated from pathological products or those that demonstrate resistance to several antibacterials. Previous works in South-Western Uganda involving reference and pathological bacteria have demonstrated huge antibac- terial resistance burden amongst HIV/AIDS patients in relation to high level re- sistance of oral bacteria associated with HIV/AIDS patients to standardised an- tibacterial discs and commercially available antibacterial agents [6] [7] [8]. However, some of the oral bacteria resisted the activity of some of the plants im- plying that antibacterial resistance is still a great public health burden in rural areas of developing nations including South-Western Uganda. This therefore calls for incisive and in-depth search into the plant world to obtain the effective and efficient antimicrobial drug entities endowed by nature to solve the present challenges of huge cost and non-availability of effective drugs, antimicrobial re- sistance, high morbidity, discomfort, extended hospitalizations, poor quality of life and possible death facing HIV/AIDS patients, and burden of poor patient management facing healthcare professionals and managers, ministries of health and governments in developing world as handlers of the challenges. 5. Conclusion and Recommendations It can be concluded that Bidens pilosa Aqueous, Bidens pilosa Ethanolic, Agera- tum conyzoides Aqueous, Ageratum conyzoides Ethanolic, Ocimum suave Aqueous and Ocimum suave Ethanolic have antibacterial activities including bacteriostatic and bactericidal potentials. These findings therefore validate the claims made by the traditional healers and local people of South-Western Uganda that Bidens pilosa Aqueous, Bidens pilosa Ethanolic, Ageratum con- yzoides Aqueous, Ageratum conyzoides Ethanolic, Ocimum suave Aqueous and Ocimum suave Ethanolic can be used to treat bacteria infected oral lesions of HIV/AIDS patients. We hence recommend an isolation, identification and structural elucidation of the bioactive antibacterial principle(s) in these plants for possible new drug entities, verification of the interactive effects of the prin- ciple(s) with ARVs and cotrimoxazole used daily by HIV/AIDS patients. Acknowledgements Authors wish to thank Mr. James Mwesigye, Department of Microbiology, Fa- https://doi.org/10.4236/pp.2017.89023 J. O. C. Ezeonwumelu et al. DOI: 10.4236/pp.2017.89023 319 Pharmacology & Pharmacy culty of Medicine of Mbarara University of Science and Technology, Mbarara, Uganda for the technical support he provided to the authors. Ethical Approval Ethical approval was pursued and acquired from The AIDS Support Organisa- tion (TASO) Kampala, Uganda National Council for Science and Technology (UNCST) and Mbarara University of Science and Technology Institution’s Re- search and Ethics Committees. These clearances allowed us to use the clinical isolates supplied by the Microbiology Department, KIU_WC. Competing Interests Authors have declared no conflicts of interest exist. Authors’ Contributions The authors collaborated in carrying out the work as follows: JOCE initiated the study. Authors JOCE, EA, AAA, TS, JCE, OED and FB collected the data. Au- thors JOCE, MN, SOO, EA, COO and FB designed the study, wrote andcor- rected the protocol.Authors KIK, JKT and JOCE wrote the protocol and thefirst draft of manuscript, searched for literature, analyzed resistance, MIC andMBC data, read through the data and made corrections.Authors MN, SOO,EA and FB managed the experimental processes, read through and madecorrections to the manuscript draft. Authors COO, TS, JCE, OED and AAA read through and made corrections to the manuscript draft. All authors read and approved the manuscript for publication. Consent Written consent was sought and secured from the Kampala International Uni- versity Microbiology Research Laboratory, Ishaka, Bushenyi where the isolates were being kept with the permission of the scientist who previously researched on these organisms. Consent to Publish The authors have given their consents to publish this article. References [1] Cowan, M.M. (1999) Plant Products as Antimicrobial Agents. 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DOI: 10.4236/pp.2017.89023 323 Pharmacology & Pharmacy Abbreviations HIV = Human Immunodeficiency Virus AIDS = Acquired Immunodeficiency Syndrome ARVs = Antiretrovirals MHA = Mueller Hinton Agar API = Analytical Profile Index MIC = Minimum Inhibitory Concentration MBC = Minimum Bactericidal Concentration MBCs = Minimum Bactericidal Concentrations MANOVA = Multiple Analysis of Variance p (p value) = Probability value KIU-WC = Kampala International University Western Campus NCCLS = National Council for Clinical and Laboratory Standards CLSI = Clinical and Laboratory Standards Institute TASO = The AIDS Support Organisation UNCST = Uganda National Council for Science and Technology. SPSS = Statistical Package for Social Sciences UTI = Urinary Tract Infections BPA = Bidens pilosa Aqueous BPE = Bidens pilosa Ethanol ACA = Ageratum conyzoides Aqueous ACE = Ageratum conyzoides Ethanol OSA = Ocimum suave Aqueous OSE = Ocimum suave Ethanol Submit or recommend next manuscript to SCIRP and we will provide best service for you: Accepting pre-submission inquiries through Email, Facebook, LinkedIn, Twitter, etc. A wide selection of journals (inclusive of 9 subjects, more than 200 journals) Providing 24-hour high-quality service User-friendly online submission system Fair and swift peer-review system Efficient typesetting and proofreading procedure Display of the result of downloads and visits, as well as the number of cited articles Maximum dissemination of your research work Submit your manuscript at: http://papersubmission.scirp.org/ Or contact pp@scirp.org https://doi.org/10.4236/pp.2017.89023 http://papersubmission.scirp.org/ mailto:pp@scirp.org In vitro Antibacterial Efficacy of Bidens pilosa, Ageratum conyzoides and Ocimum suave Extracts against HIV/AIDS Patients’ Oral Bacteria in South-Western Uganda Abstract Keywords 1. Introduction 2. Materials and Methods 2.1. Drugs and Reagents 2.2. Study Design 2.3. Sample Size and Sampling Technique 2.4. Confirmation of Bacterial Viability and Identity 2.5. Selection and Identification of Plants for Antibacterial Activity Testing 2.6. Collection, Processing and Preparation of Plant Extract 2.7. Susceptibility Testing 2.8. Broth Preparation for MIC 2.9. MIC and MBC Determination 2.10. Data Analysis 3. Results 3.1. Susceptibility of Oral Bacteria 3.2. Minimum Inhibitory Concentration (MIC) 3.3. Minimum Bactericidal Concentration (MBC) 4. Discussion 5. Conclusion and Recommendations Acknowledgements Ethical Approval Competing Interests Authors’ Contributions Consent Consent to Publish References Abbreviations