BIOCIDAL POTENTIALS OF STEM BARK EXTRACTS OF PSIDIUM GUAJAVA (LINN.) ON PANEL OF BACTERIAL STRAINS ASSOCIATED WITH DIARRHEA AND DYSENTERY

Objective: Investigations were carried out on cidal effects of crude extract and fractions obtained from stem bark of Psidium guajava against a panel of 74 strains of Escherichia coli and Shigella dysenteriae implicated in diarrhea and dysentery infections. Methods: Powdered sample of the stem bark was extracted in methanol/distilled water (3:2) and then partitioned into different organic solvents. The fractions obtained were subjected to antibacterial tests against a panel of bacterial strains. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) were determined using agar dilution method while the mode of action of the active fractions was investigated through time-kill dynamics and leakages of proteins and potassium ions from the cells. Results: All partitioned fractions except the aqueous fraction exhibited antibacterial activities against the panel of bacterial strains at a final concentration of 10 mg/mL. The MIC exhibited by the crude extract against susceptible bacterial strains ranged between 1.56 mg/mL and 12.5 mg/mL, while the MIC for the four fractions ranged between 0.31 mg/mL and 5.0 mg/mL. The MBC ranged between 3.13–12.5 mg/mL and 0.63–5.0 mg/mL for the crude extract and fractions, respectively. The time-kill assay revealed that the percentage of the cells killed increase with an increase in the concentrations of the fractions as well as contact time intervals. Proteins and potassium ions leakages from the bacterial cells followed the same trend with that of time-kill assay. Conclusion: The stem bark extracts of P. guajava exhibited appreciable bactericidal effects on bacterial strains associated with diarrhea and dysentery in humans.


INTRODUCTION
Medicinal plants serve as a source of folklore remedies for many ailments, especially among the rural dwellers in Africa and many parts of the countries worldwide. Herbal medicines are gradually becoming an integral part of primary health-care delivery. Diarrhea is a very common disease condition in most tropical communities, and the use of herbal decoction is a common practice in its management [1]. Escherichia coli and Shigella dysenteriae are the causative agents of diarrhea and dysentery, respectively. Diarrhea and dysentery are known to be killer diseases especially among the children [2,3]. These vectors are now gradually developing resistant against the available antibiotics used as therapy against infections caused by these pathogens. There is an urgent need to source for other potent antimicrobials especially of natural origin to combat infections caused by these pathogens and other microorganisms as well. This will ensure a reduction in the rate of untimely death among the children. This study thus investigated the potency of Psidium guajava stem bark extract against E. coli and S. dysenteriae. P. guajava is among the widely used medicinal plant in local folklore remedy. Its fruits and leaves have been studied for different medicinal values [4]. P. guajava belongs to the family Myricaceae and comprises 150 species. It is called apple of tropics in English and known as "Guofa" in Yoruba language. P. guajava is a large dicotyledonous shrub and is generally 3-10 m high with many branches. This tree is characterized by very thin skin and the fruit is ovoid or pear-shaped berry of about 4-12 cm long [5]. P. guajava is valued as a potential source of pectin,

Abidoye et al.
of Microbiology, Obafemi Awolowo University, Ile-Ife, Nigeria. These organisms included E. coli (NCIB 86), E. coli (ATCC 25922), S. dysenteriae (NCIB 197), and S. dysenteriae (ATCC 11311). The purity of these organisms was done before been used. The preparation of the bacterial strains was done using the colony suspension method as described by European Committee on Antimicrobial Susceptibility Testing.

Culture media used
The culture media used for this study were nutrient broth (LAB M) and nutrient agar (LAB M) for sub-culturing the bacterial strains. Mueller-Hinton agar (LAB M) was used for sensitivity testing, eosin methylene blue agar (LAB M) was used to re-confirm E. coli and S. dysenteriae, respectively. All media except Salmonella-Shigella agar were sterilized in an autoclave at 121°C for 15 min.

Collection and preparation of plant sample
The fresh stem bark of P. guajava used for this study was collected at Abeokuta, Ogun State, Nigeria, in the month of May 2015. The plant was identified in the Herbarium of Department of Botany, Obafemi Awolowo University, Ile-Ife, Nigeria. The voucher sample of the plant was prepared and deposited in the herbarium for future reference purposes with voucher number IFE-14753. The plant sample was ovendried at 40°C until a constant weight of the sample was obtained. The dried sample was later ground into a fine powder and stored in an airtight container for further use.

Extraction of biological active components of the plant sample
One thousand and five hundred grams of the powdered sample of the plant were soaked in a mixture of methanol and sterile distilled water in ratio 3:2 (v/v) and left on the laboratory bench for 4 days with regular agitation of the aliquot. The solution collected was filtered into a clean sterile flask and the supernatant collected was concentrated in vacuo using a rotatory evaporator to eliminate the methanol leaving aqueous portion. The aqueous part was then lyophilized to collect the crude extract of the plant sample.

Assessment of photochemical components in the extract from P. guajava stem bark
The assessment of the phytochemicals in the plant extract was done using Trease and Evans [16] and Harborne [17] methods. A small portion of the dry extract was screened for the presence of alkaloids, tannins, steroids, cardiac glycosides, reducing sugars, and saponins.

Fractionation of the crude extract
The crude extract was fractionated using a different organic solvent in order of their polarity, starting with low polar solvent and graduated to the highest polar solvent that is, starting with n-hexane and finished (contd...)

Bacterial isolates
Psidium guajava (   up with n-butanol. The fractions obtained were kept in an air-tight container in a refrigerator at 5°C for further use.

Determination of the antibacterial potency of the crude extract and fractions obtained from it
The antibacterial tests were done using agar-well diffusion methods [18]. The test organisms were re-activated in nutrient broth for 18 h before use. Exactly 0.1 ml of standardized test bacterial strains (10 6 cfu/mL of 0.5 McFarland standard) was transferred into Mueller-Hinton agar medium at 40°C. This was thoroughly mixed together and later poured into pre-sterilized Petri dishes. The plates were allowed to set and wells were bored into the medium using 6 mm sterile cork borer. These wells were then filled up with the prepared solutions of the crude extract and the fractions. Care was taken not to allow the solution to spill on the surface of the medium. The final concentrations of the crude extract and the fractions used were 25 mg/mL and 10 mg/mL, respectively. On the other hand, ampicillin and streptomycin were used     as positive controls at a concentration of 1 mg/mL. The plates were left on the laboratory bench for 1 h to allow proper in-flow of the solution into the medium before incubating them at 37°C for 24 h. The plates were not stock-piled to allow even distribution of temperature round the plates to avoid false results. The plates were later observed for zones of inhibition which is an indication of susceptibility of the test organisms to the extracts.

Assessment of minimum inhibitory concentrations (MIC) of the crude extract and fractions against susceptible organisms
Two-fold dilution of the crude extract and fractions was adopted to determine their MIC [19]. Two milliliters of different concentrations of the solution were added to 18 mL of pre-sterilized molten nutrient agar to give the final concentrations ranging between 0.31 mg/mL-5.00 mg/mL for the fractions and 1.56 mg/mL-12.5 mg/mL for crude extract. The mixture was then poured into well-labeled sterile Petri dishes and allowed to solidify. The plates were left on the laboratory bench overnight to ascertain their purity. The surfaces of the plates were allowed to dry before streaking with the standardized inoculums of the organisms and incubated aerobically at 37°C for 48 h. The plates were later examined for the presence or absence of bacterial growth. The MIC was taken as the lowest concentration of the fraction that inhibited the growth of the organisms.

Assessment of minimum bactericidal concentration (MBC) of the crude extract and fractions against susceptible organisms
The MBC of the fractions and the crude extract was assessed by taken sample from streaked line of the MIC test and cultured on fresh sterile nutrient agar medium. The plates were incubated at 37°C for 72 h. The MBC was taken as the concentrations of the fraction that did not support any bactericidal growth of the medium.

Determination of the killing rate of bacterial strains by the fractions
The test was carried out using test bacterial strains, that is, E. coli and S. dysenteriae on their viability against different concentrations of fractions relative to their MIC [18]. Viable counts of the test organisms were initially determined to standardize the inoculums. Exactly 0.5 mL volume of unknown cell density (by viable counts 10 6 cfu/mL) was added to 4.5 mL of different concentrations of the fraction relative   [20]. The standard curve for protein analysis was constructed to quantify the quality of protein leaked from the bacterial cells. Exactly 0.4 mL Bradford reagent was added to 1.6 mL sample (0.2 mL supernatant added to 1.4 mL sterilized distilled water) to make up 2 mL total volume. Optical density (OD) of the resulting solution was thereafter taking at 595 nm after 5 min of preparation but not later than 1 h. The OD of each of the samples was calculated from the equation of the best-fit linear regression line obtained from the graph of the bovine serum albumin (BSA) standard curve.

Preparation of the BSA standard curve for the quantification of protein leaked out of the bacterial cells
Exactly 100 ug/mL concentration of BSA stock solution was prepared and varying concentrations were made from the stock solution. A 0.4 mL of Bradford reagent was added to the various BSA concentrations. This to MIC. The suspension was thoroughly mixed and held at room temperature (28-30°C) for 2 h to determine the killing rate of the test organisms. Exactly 0.5 mL of each suspension was withdrawn at the appropriate time intervals and transferred to 4.5 mL nutrient broth recovery medium containing 3% "Tween 80" to neutralize the effects of the fractions carry-over from the bacterial cells. The suspension was shaken properly and then serially diluted up to 10 −5 in sterile physiological saline and plated out for viable counts. The plates were incubated at 37°C for 48 h before determining the survival cells. Control experiment was set up without the inclusion of the fractions. Viable counts were made in triplicates for each sample and compared with the counts of the control. Depression in viable counts indicated killing by the fractions.

Determination of protein leakage from the bacterial strains by the fractions
The test bacterial strains cells were (E. coli and S. dysenteriae) separately washed in 0.9% (w/v) normal saline to remove any presence of slime layer from the cells. These washed cells (inoculum size

Determination of potassium ions leakage from the bacterial cells by the fractions
Eighteen-hour old cells of E. coli and S. dysenteriae were washed 3 times in physiological saline to remove slime layers from the cells. Exactly 50 mL of washed cells (OD 470 nm=1.5) was dispensed into a clean beaker which was magnetically stirred. Fifty milliliters of ionic strength adjustment buffer (18.37 g of tetraethylammonium chloride in deionized water and made up to 100 mL) were added to the beaker to make the background ionic strength of all solution kept constant. The potassium ion sensing electrode (Qualiprobe QSE 314, EDT instruments Waldershare Park, Dover, UK) and its reference electrode (Qualiprobe double junction reference electrode E 8092 EDT instruments) were placed into the cell suspension. The potential difference (mV) derived by the electrodes was measured using a Whatman Maidstone, UK. E. coli and S. dysenteriae cells were treated with various concentrations of the fractions relative to the MIC. The potassium ions leaked out from the cells of the organisms were measured at time interval over a period of 2 h as a potential difference in mV. These values were later converted to concentrations of potassium ions by reference to a conversion graph which had been constructed using potassium chloride standard solutions. The concentrations of potassium ions released were plotted against time.

Statistics and data processing
The experiments were carried out in triplicates. Data were analyzed by a 4×4 Latin square designated with the statistical program using the GLM model (Statistical Analysis Systems Institute, Cary NC, USA, 2001). Results were contrasted with negative and a positive control. The mean of the values was compared using independent t-test of significance (p<0.05).

RESULTS
Exactly 130.5 g of crude extract was obtained from 1500 g of powdered stem bark of P. guajava. This gave 8.7% yield of the total weight of powdered sample used and the extract was dark brown in color. Four fractions were obtained from the crude extract and these include n-hexane, chloroform, ethyl-acetate, and butanolic fractions. The crude extract along with the four fractions exhibited appreciable antimicrobial activities against all the 72 bacterial strains used for this study. The crude extract exhibited antimicrobial activity at a final concentration of 25 mg/mL while all the four fractions exhibited antimicrobial activities at a final concentration of 10 mg/mL. On the other hand, streptomycin at a concentration of 1 mg/mL inhibited the growth of 64 test organisms while ampicillin at the same concentration with streptomycin inhibited the growth of 22 organisms only (Tables 1 and 2). Overall, the crude extract along with the four fractions compared favorably with the two The MIC and MBC of the crude extract along with those of the four fractions were also assessed. The crude extract and the fractions exhibited varying degrees of MIC and MBC against test organisms used for this study, as indicated in Tables 3 and 4. The MIC exhibited by the crude extract against both E. coli and S. dysenteriae ranged between 1.56 mg/mL and 12.5 mg/mL. On the other hand, the range exhibited by the fractions against susceptible organisms ranged between 0.31 mg/mL and 5.0 mg/mL.
The MBC exhibited by the crude extract against the bacterial strains followed the same trend with those observed for the MIC. The MBC ranged between 3.13 mg/mL and 12.5 mg/mL. On the other hand, the MBC observed for the fractions against E. coli and S. dysenteriae ranged between 0.63 mg/mL and 5.0 mg/mL.
The phytochemical compounds that were responsible for the biological activities of the stem bark extract of P. guajava were also investigated as shown in Table 5. The extract revealed the presence of saponins, alkaloids, flavonoids, tannins, steroids, cardiac glycosides, and reducing sugars.
The biocidal potentials of the fractions obtained from the stem bark extract of P. guajava were also investigated. This was assessed through the killing rate effects of the test cells protein and potassium ions leakages from the test cells. Fig. 1a    S. dysenteriae cells were also subjected to the effects of the fractions for potassium ions leakage. Appreciable quantities of potassium ion were also leaked out of the cells and follow the same trend with those observed for the E. coli cells. Fig. 6a-c shows the effects of the four fractions on S. dysenteriae cells. The higher the concentrations of the extract and contact time, the more potassium ions got leaked out of this bacterial cells as observed for E. coli.