The plant material consisted of the leaves, stems, and roots of Phyllanthus muellerianus, collected at Amoussimé in Tabligbo, a village situated about 79 km from Lomé (Togo). The plant was identified and confirmed at the Botany and Plant Ecology Laboratory of the Faculty of Science at the University of Lomé. The organs of the plant are shown in Figure 1(a) and Figure 1(b) .

The bacterial strains are made up of clinical and reference strains. The reference strains: Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 13883 were obtained from the bacteriology laboratory of the National institute of hygiene in Lomé. Clinical strains of S. aureus (S. aureus 0454; S. aureus 066; S. aureus 064; S. aureus 063, S. aureus 062; S. aureus 056), Staphylococcus spp. (Staphylococcus spp. 054; Staphylococcus spp. 0747; Staphylococcus spp. 0746; Staphylococcus spp. 048), Escherichia coli (E. coli 0913; E. coli 068; E. coli 476), Pseudomonas aeruginosa (P. aeruginosa 061; P. aeruginosa 065; P. aeruginosa 070) and Klebsiella pneumoniae (K. pneumoniae 0606; K. pneumoniae 823; K. pneumoniae 452) were obtained from the bacteriology laboratory of the Lomé Campus university hospital. Clinical strains were isolated from urine and pus samples and chosen in this study for their resistance against some reference drugs.

The leaves, stems and roots of P. muellerianus were washed and finely cut, then dried in the laboratory under air conditioning for two weeks. The plant organs were then ground to a powder using an electric grinder. For each plant part, hydroethanolic extracts were prepared following the method used by Hoekou et al. The hydroethanolic extract was obtained by macerating 50 g of powder in 500 mL of ethanol-water (70/30 (v/v)) 30˚C for 48 hours under 300 rpm of stirring. This ethanol-water ratio is better for extracting the polar and nonpolar compounds contained in the plant organ and is compatible with the recommendations of traditional healers. The macerate was filtered through Whatman paper No.1. The various filtrates obtained were evaporated in a Rotavapor (Buchi type) and then freeze-dried to obtain dry extracts [19] . The yield of the different extractions was calculated according to Equation (1):

$Yield\left(g\cdot \frac{100}{g}\right)=\frac{Weightofdryextract\left(g\right)}{Weightofplantpowder\left(g\right)}\times 100$ (1)

The various clinical and reference strains were grown on nutrient agar for 18 - 24 hours. A characteristic colony of each strain was taken by sterile loop and inoculated into 10 mL of sterile physiological water. A turbidity solution equivalent to 0.5 Mc Farland was used to adjust the different microbial suspensions prepared. One gram of each lyophilised extract is dissolved in 10 mL of a sterile ethanol/distilled water mixture to obtain a solution with a concentration of 100 mg/mL, and it was diluted to obtain a concentration of 40 mg/mL. The extracts were sterilised using a syringe filter on 0.22 nm Millipore membrane and verified by culturing an aliquot of this solution on Mueller Hinton agar for 24 hours at 37˚C in an incubator.

The agar diffusion method with antibiotic-impregnated discs was used. The inoculums were prepared from a pure culture of 18 to 24 hours. To do this, a colony of this pure culture was suspended in 10 mL of saline solution (0.9% NaCl), then the suspension obtained was adjusted by comparison with the 0.5 Mac Farland control. The inoculum thus prepared was used to inoculate Mueller Hinton agar. Antibiotic discs were applied to the agar, which had previously been inoculated with the strain to be tested. After pre-diffusion for 15 minutes at laboratory temperature, the plates were then incubated at 37˚C for 18 to 24 hours. The diameter of the inhibition is measured using a ruler (cm) and then compared with the concordance scale for each antibiotic supplied. This diameter allows to categorise the strains as sensitive (S) or resistant (R) to each antibiotics tested [20] .

The agar well diffusion method was used to assess the antimicrobial properties of different extracts [21] [22] . The prepared microbial suspensions were used to inoculate Mueller Hinton agar by flooding. The tips cut to an internal diameter of 6 mm and then sterilized were used to make wells in the agar. 50 µL of the different hydroethanolic extracts (40 mg/mL) were deposited in each well. Ethanol-distilled water mixture and gentamicin (5 µg/mL) were used as negative and positive controls respectively. After the pre-diffusion of the extract, all the plates were then incubated at 37˚C for 24 hours. The antibacterial susceptibility was assessed by measuring the inhibition diameters around the wells. All tests were replicated three times.

The MIC and MBC were determined using the microdilution technique with Mueller Hinton (MH) broth [22] . A serial dilution of the different hydroethanolic extracts were made from 20 mg/mL to 0.625 mg/mL and 100 µL of each dilution were dispensed in the microplate containing 100 µL of the different bacterial strain suspensions. The microplates were incubated at 37˚C for 24 hours. The MIC was recorded as the minimum concentration of extract for which no visible growth was observed. The MBC is determined by removing 100 µL of suspension from wells with no visible growth and inoculating nutrient agar plates that were incubated at 37˚C for 24 hours, after which the strain colonies were counted. The MBC/MIC ratio was used to assess the activity of the extract.

The main phytochemical groups in plant extracts were identified by means of a summary qualitative phytochemical analysis based on colour tests [23] . Tannins, flavonoids, alkaloids and saponins were sought using two methods.

Tannins: Three drops of 1% FeCl₃ solution were added to 1 ml of extract and incubated for two minutes. Or 1 mL of lead acetate solution at 10% was added to 3 mL of extract and incubated for 2 minutes. A blue-black coloration indicates the presence of tannins in two tests.

Flavonoids: Two drops NaOH of 1% solution were added to 1 mL of extract; a positive test was revealed by the presence of a yellow coloration. Or 2 mL of extract are treated with 3 drops of 1% FeCl₃ solution; the positive test is marked by the appearance of a green coloration.

Alkaloids: Two drops of Dragendorf or Mayer reagent are added to 2 mL of extract. A positive test was marked by a red or white coloration respectively.

Saponins: 1 mL of distilled water was added to 2 mL of aqueous extract, then the solution is stirred for 1 minute. The saponins were confirmed by the presence of a foam that persists for 15 minutes and exceeds 1 cm of height.

Total polyphenols are measured using the Folin-Ciocalteu Reagent method [24] . The total phenol content is determined by extrapolation on a standard curve obtained from a series of dilutions with distilled water of gallic acid (200 mg/L) ranging from 0.1 to 0.25 mg/mL. A mixture consisting of 0.2 mL of 1 mg/mL extract and 0.5 mL of FCR diluted at half in distilled water was added to the test tubes. After 5 minutes incubation at room temperature, 0.5 mL sodium carbonate (20 g/L) was added to the mixture. The volume in each tube was made up to 4 mL. After shaking, the different solutions were left to stand, protected from light, for 30 min. The optical density was read at 760 nm using a visible UV spectrophotometer (UNICO model 12) against a negative and positive blank. Three readings were taken per sample, and the total phenol content of the various extracts was determined by the following Equation (2).

$TP=\left(\frac{mgAGE}{g}\right)=\frac{\left(OD-0.0218\right)}{0.2074}$ (2)

where OD is the optical density measured at 760 nm, TP (mgAGE/g) is the equivalent gallic acid concentration per gram (mgAGE/g).

Condensed tannin content was assessed using the Butanol-HCl method [25] . The reaction leads to the release of anthocyanidins corresponding to the cleaved monomers, which absorb at 540 nm. The test consisted of adding 0.2 mL of ammoniacal iron sulphate (20 g/L) and 7 mL of a butanol/HCl solution (95/5 mL) to 0.2 mL of each extract in tubes. After 30 min incubation in a water bath (Biobase type) at 95˚C, the tubes were cooled and the absorbances were read at 540 nm. The concentration of condensed tannins in the extracts was obtained using the following Equation (3) [26] .

$CT=\left(\frac{mgCE}{g}\right)=\frac{\left(OD\times 1CE/g\right)}{0.280}$ (3)

where OD, is optical density measured at 540 nm; 1 CE/g, represent catechin equivalent; CT (mgCE/g) is the catechin equivalent content per gram (mgCE/g).

Phosphomolybdate reduction was carried out using the method described by Prieto et al. (1999); the reaction is based on the reduction of molybdate (VI) to molybdate (V) by the reducing compounds present in the extract after incubation for 90 min at 95˚C. The reduced compound is measured with a spectrophotometer at 695 nm. Phosphomolybdate (100 mL) was prepared from a mixture of 90 mL 0.6 M sulphuric acid, 5 mL 0.1% sodium phosphate and 5 mL 1% ammonium molybdate. To carry out the test, 1 mL of each extract was added to 9 mL of phosphomolybdate. The whole mixture was heated in a water bath at 95˚C for 90 min. Absorbance was measured at 695 nm. Ascorbic acid was used as the standard antioxidant [27] . The ascorbic acid content was determined by the following equation (4).

$FRA=\left(\frac{mgAAE}{g}\right)=\frac{\left(OD\times 0.2222\right)}{0.0049}$ (4)

where OD is the Optical Density measured at 695 nm: FRA (mgAAE/g) represent the equivalent ascorbic acid concentration per gram (mgAAE/g).

All experiments were performed in triplicate and mean values are presented with standard deviation (mean ± SD).

The yield of hydroethanolic extracts from the three plant organs are shown in Figure 2 . From 50 g of leaf, stem and root powders, the extract yields were 21.99%, 5.94% and 3.66% respectively. The extract from the leaves of P. muellerianus showed the highest extraction yield.

Reference and clinical strains were tested with various reference antibiotics. The sensitivity profile of the different germs is shown in Table 1 . Analysis of this table shows that S. aureus (n = 6), Staphyloccocus spp. (n = 4), Escherichia coli (n = 3), Pseudomonas aeruginosa (n = 3) and Klebsiella pneumoniae (n = 3) are resistant to several of the antibiotics tested, including Ampicillin, Amoxicillin, Ticarcillin, Piperacillin and Cefalotin.

NT: not tested, S: sensitive, R: resistant.

The sensitivity of germs to the different extracts is presented in Figure 3 . Hydroethanolic extracts of leaves (PmF), stems (PmT) and roots (PmR) were active on S. aureus (n = 6), Staphyloccocus spp. (n = 4) and S. aureus ATCC29213 with inhibition diameters ranging from 15 to 23 mm. PmF and PmR were active on P. aeruginosa (n = 3) and P. aeruginosa ATCC27853 with inhibition diameters ranging from 10 to 15 mm. PmF was active on K. pneumoniae ATCC 1388 and E. coli ATCC 25922 with inhibition diameters of 10 mm and 11 mm respectively. K. pneumoniae (n = 3) and E. coli (n = 3) were resistant to the three P. muellerianus extracts tested in this study. In the Gram-positive bacteria tested, PmF was more active than PmT, which was also more active than PmR (PmF > PmT > PmR). For the Gram-negative bacteria tested, only PmF and PmT showed activity with Pseudomonas strains. In this study, the three hydroethanolic extracts were tested at 40 mg/mL. Gentamicin (reference drug tested at 5 µg/mL), used as a positive control, was active on all germs with inhibition diameters ranging from 10 to 22 mm. The negative control is inactive on all germs.

In this section, MICs were determined for the hydroethanolic extract of P. muellerianus leaves on Staphylococcus strains (n = 10); PmF showed better activity than PmT and PmR, with staphylococci strains. The results are presented in Table 2 . The MBC and MBC/MIC ratio were also determined. The MIC and MBC values obtained ranged from 2.5 to 10 mg/mL. The best MICs and MBCs were obtained with Staphylococcus spp. 048 and Staphylococcus spp. 0746 with a value of 2.5 mg/mL. The MBCs determined were the same as the MICs for the extract on the tested germs. The MBC/MIC ratio was therefore unit. The interpretation of this MBC/MIC ratio allows us to conclude that PmF has bactericidal activity on the Staphylococcus strains used.

PmF: Hydroethanolic extract of P. muellerianus leaves, MIC: minimum inhibition concentration, MBC: minimum bactericidal concentration.

The phytochemical study revealed the major phytochemical groups contained in the various extracts of P. muellerianus. The tests were carried out in duplicate using two different methods, except in the case of saponins. The results are summarised in Table 3 . The three extracts contain flavonoids, alkaloids, saponins and tannins.

+: Presence; -: Absence; PmF: hydroethanolic extract of leaves of P. muellerianus; PmT: hydroethanolic extract of stem of P. muellerianus; PmR: hydroethanolic extract of root of P. muellerianus.

The phytochemical screening enabled us to highlight the presence of groups of bioactive molecules. It would be very interesting to measure these compounds and determine their activity. Total polyphenolic and condensed tannins were measured to assess the total anti-free radical activity of these compounds. The results are shown in Figures 4-6 .

Total polyphenolic in the three extracts was shown in Figure 4 . PmF extract has a higher content than PmT and PmR, with a value of 4.23 ± 0.25 mgAGE/g. The condensed tannin content (CT) was evaluated in the three extracts and shown in Figure 5 . The extract of stems of P. muellerianus (PmF) has the highest content with a value of 2.97 ± 0.05 mgCE/g. Total anti-free radical activity (fRA) is shown in Figure 6 . This activity was highest with the root extract (PmR), which was 0.125 ± 0.003 mgAAE/g.