{"id":54771,"date":"2023-12-31T11:48:51","date_gmt":"2023-12-31T11:48:51","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=54771"},"modified":"2024-01-05T05:55:18","modified_gmt":"2024-01-05T05:55:18","slug":"antibacterial-activity-of-crude-aqueous-extracts-of-tithonia-diversifolia-from-chichiri-area-in-blantyre-district-malawi","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol16no4\/antibacterial-activity-of-crude-aqueous-extracts-of-tithonia-diversifolia-from-chichiri-area-in-blantyre-district-malawi\/","title":{"rendered":"Antibacterial Activity of Crude Aqueous Extracts of Tithonia Diversifolia from Chichiri Area in Blantyre District, Malawi."},"content":{"rendered":"\n

Introduction <\/strong><\/p>\n\n\n\n

Various studies have reported alarming trends in antimicrobial\nresistance in blood stream infection isolates in Malawi1, 2, 3<\/sup> and an\nongoing large scale study is characterizing the morbidity, mortality and\neconomic cost of third-generation cephalosporin resistant bloodstream infection\n4<\/sup>.<\/p>\n\n\n\n

The combination of restricted access to the few effective antibiotics in\nresource-poor settings such as Malawi and the global burden of AMR has\nspurred scientific investigation of phytochemicals as an alternative source of\nnew antimicrobial drugs. <\/p>\n\n\n\n

Medicinal plants in most developing countries, including Malawi, are well recognized as alternative therapeutic agents for the maintenance of good health5<\/sup>. In an ethnobotanical study of traditional medicinal plants used for the treatment of infectious diseases by local communities in Mzimba district of the northern region of Malawi, Chisamile recorded eighty medicinal plants belonging to 43 families and 77 genera6<\/sup>. Similarly, in an ethnomedicinal survey by Chikowi in Zomba district of the southern region of Malawi, fifty-nine medicinal plant species belonging to 38 families were reported to be in use as prophylaxis and treatment for 27 communicable and non-communicable diseases\/conditions7<\/sup>. The afore-cited research findings underscore the observation that Malawi has a rich biodiversity of medicinal plant species that represent an exploitable resource in discovery research for lead bioactive compound development.<\/p>\n\n\n\n

There is, however, a paucity of published data from Malawi on the in\nvitro<\/em> antibacterial\nactivity of native\nmedicinal plants. Pterocarpus angolensis<\/em> (locally known as Mlombwa tree) grows in many parts of Malawi. In a study by Chipinga, the aqueous,\ndichloromethane and methanolic extracts of the leaves, stem-bark, fruits and\nroots of Pterocarpus angolensis<\/em>\nwere shown to be effective against Escherichia coli, Staphylococcus\naureus, Streptococcus agalactiae and Candida krusei<\/em> by the macrotube\ndilution method.<\/p>\n\n\n\n

Tithonia diversifolia<\/em> (Hemsl) A. Gray is a member\nof the sunflower family, Asteraceae. Whereas this plant is native to\nCentral America and the West Indies, it has become naturalized in Malawi,\ngrowing around agricultural fields, in waste places, along\nriver banks and in many other ecological environments. Decoctions or infusions\nof T. diversifolia<\/em> have been\nwidely reported as being of medicinal use in many countries:\ndemonstrating antibacterial, antiplasmodial activities of various parts of the plant8-11<\/sup>.\nExternal use on wounds has also been reported12<\/sup>. The\nethnopharmacological importance of this plant is comprehensively reviewed by\nAjao and Moteetee13<\/sup>.<\/p>\n\n\n\n

It is recognized that Tithonia diversifolia<\/em> can grow in many different environmental conditions14<\/sup> where it does not require a large amount of nutrients because it is able to increase the amount of essential nutrients in the soil on its own15<\/sup> and exhibits seasonal as well as geographical variation in phytochemical composition16,17<\/sup>. Geographical variation in phytochemical composition has been observed in other plant species18,19<\/sup>.   In the present study the purpose was, therefore, to investigate and validate the in vitro <\/em>antibacterial activity of aqueous extracts of T. diversifolia<\/em> growing in Blantyre city, Malawi.<\/p>\n\n\n\n

Materials and Methods<\/strong><\/p>\n\n\n\n

Nutrient agar (ATICO India, India), Mueller-Hinton agar (Oxoid, UK), Blood Agar Base (Mast Group Ltd, UK), Bacterial isolates (E. coli, Proteus mirabilis, K. pneumoniae) from Malawi-Liverpool Wellcome Trust Program, drugs (Malawi Medicines Regulatory Authority).<\/p>\n\n\n\n

Source of Samples, Identification, and Preparation<\/strong><\/p>\n\n\n\n

This study was conducted from the 16TH<\/sup> of November, 2022 to the 6TH<\/sup> of December, 2022. T. diversifolia<\/em> (Figure 1) samples were collected from Chichiri near Polytechnic hostels and transported in a cooler box to the Biological Sciences Laboratories at The Malawi University of Science and Technology (MUST).  T. diversifolia<\/em> specimens underwent identification and authentication by the National Herbarium and Botanic Gardens of Malawi and the voucher specimens (Accession number 84191) were deposited at the herbarium. Once at MUST, plant samples (the leaves, roots and stems) were washed with distilled water and left to air dry separately for 4 days away from sunlight as shown in the setup in Figure 2A.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1<\/strong>: Photograph of T. diversifolia<\/em> from Chichiri area,
Blantyre, Malawi.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Plant Extraction Procedure<\/strong><\/p>\n\n\n\n

The air-dried leaves, stems and roots of T. diversifolia <\/em>were crushed separately using traditional mortar and pestle typically used by ordinary Malawians when preparing these plants for medicinal use (Figure 2B). This resulted in a fine powder of each plant part as shown in Figure 2C. <\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 2<\/strong>: Photographs showing preparation of T. diversifolia<\/em>. (A) Air-drying of plant. (B) Powdering of dry plant material using traditional mortar and pestle. (C) Tithonia powder by plant part.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Thirty grams (30 g) of each powder obtained from the\nleaves, stems and roots was separately suspended in 225mls of sterile distilled\nwater in three conical flasks which were cupped to be airtight. The suspension\nof the roots, leaves and stem were then left to soak in the airtight conical\nflasks for 2 days before filtration into a Petri dish using a cheese-cloth to\nobtain the desired aqueous filtrate for each plant part. The filtrate was then\ntransferred from the Petri dish to an evaporation flask using a sterile funnel.\nGentle evaporation of the filtrate was done in a Pyrex glass beaker by using a heating mantle sent at 450<\/sup>C until a\nresidue was obtained. <\/p>\n\n\n\n

Test Organism<\/strong><\/p>\n\n\n\n

The clinical\nisolates of bacteria (Klebsiella pneumoniae, Escherichia\ncoli and Proteus spp.<\/em>)\nused in this studywere provided by\nThe Malawi Liverpool Welcome Trust laboratory based at Queen Elizabeth Central Hospital in Blantyre and were stored in the freezer at -800<\/sup>C. <\/p>\n\n\n\n

Preparation of Nutrient Agar<\/strong><\/p>\n\n\n\n

Nutrient Agar was prepared according to the\nmanufacturer\u2019s recommendation which was to suspend 14g of the nutrient agar\npowder in 500mls of distilled water. The suspension was left on a heating\nmantle at 1000<\/sup>C for 6 minutes to completely dissolve the powder\nwhich was later autoclaved at 1210<\/sup>C and a pressure of 15psi for 15\nminutes. The prepared media was then poured into 17 sterilized petri dishes\naseptically and left to solidify for 20 minutes. The prepared media was left in\nthe fridge for storage. <\/p>\n\n\n\n

Preparation of Mueller-Hinton Agar<\/strong><\/p>\n\n\n\n

Mueller Hinton Agar was prepared according to the manufacturer\u2019s recommendation which was suspending 38g of the Mueller Hinton agar powder in 1000mls of distilled water. The suspension was left on a heating mantle at 1000<\/sup>C for 6 minutes to completely dissolve the powder which was later autoclaved at 1210<\/sup>C and a pressure of 15psi for 15 minutes. The prepared media was then poured into 30 sterilized petri dishes aseptically and left to solidify for 20 minutes. The prepared media was left in the fridge for storage.<\/p>\n\n\n\n

Preparation of Blood Agar (BA)<\/strong><\/p>\n\n\n\n

Blood Agar was prepared according to the manufacturer\u2019s recommendation which was suspending 9.375g of the nutrient agar powder in 250mls of distilled water. The suspension was left on a heating mantle at 1000<\/sup>C for 6 minutes to completely dissolve the powder which was later autoclaved at 1210<\/sup>C and a pressure of 15psi for 15 minutes. After autoclaving, the suspension was left to cool to 450<\/sup>C were 5% of human blood was added. The prepared media was then poured into 4 sterilized petri dishes aseptically and left to solidify for 20 minutes. The prepared media was left in the fridge for storage.<\/p>\n\n\n\n

Bacterial culture<\/strong><\/p>\n\n\n\n

E. coli<\/em> and K. pneumoniae<\/em> isolates were resuscitated by streaking on separate petri dishes of nutrient agar and incubating at 370<\/sup>C for 24 hours. Proteus <\/em>was resuscitated on blood agar at 370<\/sup>C for 24 hours.  Stock cultures of the resuscitated E. coli<\/em>, K. pneumonia<\/em> and Proteus<\/em> isolates were then maintained at 4\u00b0C on slopes of nutrient agar and blood agar respectively.<\/p>\n\n\n\n

Preparation of inoculum<\/strong><\/p>\n\n\n\n

Bacterial (Klebsiella pneumoniae, Escherichia coli and Proteus spp<\/em>)\ninoculums were prepared with Nutrient agar and Blood Agar and standardized to\n0.5 McFarland solution. 1 in 100 dilutions of the standardized Nutrient and\nBlood agar preparations brought the cell count to 5\u00d7106 <\/sup>CFU\/ml which\nrepresented inoculum stocks. <\/p>\n\n\n\n

Antimicrobial Susceptibility Testing<\/strong><\/p>\n\n\n\n

Antibacterial activity by disc diffusion assay<\/strong><\/p>\n\n\n\n

The\nKirby-Bauer disc diffusion assay was carried\nout based on the Clinical Laboratory Standard Institute (CLSI)\nguidelines. To\nevaluate the drug\nsusceptibility of study bacterial isolates, drug-impregnated disks of common\nantibiotics (Chloramphenicol, Gentamicin, Sulfamethoxazole Trimethoprim,\nAmoxicillin and Erythromycin) were placed on 3 separate agar plates inoculated\nwith either E. coli <\/em>or K. pneumoniae<\/em> or P. mirabilis<\/em>. In these\ntests blood agar plates were used for P. mirabilis <\/em>while Mueller Hinton agar plates were used for E. coli <\/em>and K. pneumoniae.<\/em><\/p>\n\n\n\n

To evaluate the activity of\nthe plant extracts against the bacterial isolates, disks (6 mm in diameter)\nimpregnated by different concentrations of the plant extracts (0.625g\/ml,\n0.333g\/ml, 0.165 g\/ml, 0.083g\/ml, 0.041g\/ml and 0.021 g\/ml), Gentamicin (10\u00b5g as positive\ncontrol) and a blank disk (negative control) were placed on each of the agar plate\nthat had been inoculated with a test isolate. All the plates were then incubated\nfor 24 hours at 370<\/sup>C. <\/p>\n\n\n\n

Antimicrobial\nactivity was evaluated by measuring the zones of inhibition, against the tested\nmicroorganism in millimeter (mm). Each assay was carried out in duplicates.<\/p>\n\n\n\n

Antibacterial Activity by Broth Macrodilution Assay<\/strong><\/p>\n\n\n\n

For\neach serial dilution of the plant extracts (concentrations: 62.5 mg\/ml, 33.3 mg\/ml, 16.7\nmg\/ml, 8.33 mg\/ml, 4.17 mg\/ml, 2.08 mg\/ml, 1.04 mg\/ml, 0.52 mg\/ml, 0.26 mg\/ml,\n0.13 mg\/ml) a final bacterial cell count of about 5 x 105<\/sup>\nCFU\/mL was achieved by transferring 1\nml of the prepared inoculum for each microorganism into the appropriate volume\nof serial dilution of each extract, representing 1 in 10 dilution of the\nrespective inoculum stock. After\n24 hours of incubation at 370<\/sup>C, bacterial cells were enumerated by direct microscopic\ncount method.<\/p>\n\n\n\n

Data management and analysis<\/strong><\/p>\n\n\n\n

After 24 hrs of incubation some Typical photos of agar plates were taken to demonstrate typical inhibition zones against the bacterial strains. Estimates of zones of inhibition are tabulated in MS Word based on plant extract concentration level versus bacterial strain. For each plant extract (i.e. for root extract or stem extract or leaf extract) and commercial antibiotic disks, inhibition zone diameters are reported in the tables. Inhibition zone data at 0.625 g\/ml of extract or Gentamicin was pooled from all the tabulated data and used to calculate Mean (+\/-SD)[a1]  and 95% Confidence Interval for the inhibition zone estimates and reported within the text of the results section. The Mean(+\/-SD) calculations were done using Stata SE version 17.0 (Stata Corp, College Station, TX, USA). <\/p>\n\n\n\n

Broth macrodilution assay data were analyzed in GraphPad Prism 8.0 (https:\/\/www.graphpad.com) and results are reported as graphs of percentage of surviving bacterial cells versus concentration of crude T. diversifolia<\/em> extract. From these graphs the minimum concentration (of plant extract) that inhibits 50% of the bacterial strain (MIC50) can be read off.<\/p>\n\n\n\n

Results<\/strong><\/p>\n\n\n\n

Growth Inhibition Zone <\/strong><\/p>\n\n\n\n

Tables 1-3 show measured (after 24 hours of incubation) clear zones of inhibition around the drug-impregnated disks. Based on the EUCAST breakpoint interpretation for the antibiotic\u2019s zone of inhibition all the isolates, K. Pneumoniae<\/em>, P. mirabiris<\/em> and E. coli<\/em> were sensitive to Gentamicin with mean inhibition zone ranging from 22 \u2013 25 mm (23\u00b1 1.5 mm). For this reason, Gentamicin was chosen for use as the positive control antibiotic in both the Kirby-Bauer disc diffusion and broth macrodilution assays for the assessment of Tithonia diversifolia <\/em>antibacterial potency. <\/p>\n\n\n\n

Tables 4-6\nshow inhibition zone data that demonstrate antibacterial activity of crude aqueous\nextracts (leaf, stem and roots) of Tithonia\ndiversifolia<\/em> by the Kirby-Bauer disc diffusion assay. At a concentration of\n0.625g\/ml, the mean inhibition zone for the leaves, stems and root extracts (mean\n= 18.0 \u00b1 0.9 mm, 95%CI: 16.0 – 20.0 mm, n=12) were comparable to inhibition\nzone diameters for Gentamicin (Mean = 17.3 \u00b1 0.9 mm, 95%CI:\n15.9 – 18.6 mm, n=12) action against E.\ncoli<\/em> and K. pneumoniae<\/em> only. In\nthe same concentration, Tithonia\ndiversifolia<\/em> leaf, stem and root extracts appeared significantly less\ninhibitory towards P. mirabilis<\/em> when\ncompared to Gentamicin (mean zone diameter = 13.7 \u00b1 1.5 mm, 95% CI: 12.1 \u2013 15.2 mm, n=6 versus zone diameter =23.3\n\u00b1 3.7 mm, 95% CI: 19.5 \u2013 27.2 mm, n=6 respectively; p=0.0003). Figures 3 \u2013 5 show typical agar plate pictures of the Kirby-Bauer disc diffusion assay results. <\/p>\n\n\n\n

Generally,\nbased on estimated diameters of inhibition, leaf extracts of Tithonia diversifolia<\/em> had significantly\ngreater antibacterial activity (19.5 \u00b1 3.9 mm, 95% CI: 15.4 –   23.6 mm) than stem (15.2 \u00b1 2.0 mm, 95% CI:\n13.0 – 17.3 mm, p = 0.021) or root extracts (15.0 \u00b1 2.1 mm, 95% CI: 12.8 – 17.2\nmm, p=0.019).<\/p>\n\n\n\n

When\nassayed against P. mirabilis<\/em>, the leaf extract mean inhibition zone diameter (15.5 \u00b1\n0.5 mm [95%CI: 9.1 – 21.9 mm]) was significantly wider than the stem extract\nzone diameter (13.0 \u00b1 0.0 mm [95% CI: 13.0 \u2013 13.0 mm], p=0.02) whereas when\ncompared to the root extract inhibition zone it tended towards being wider\nwithout achieving statistical significance (12.5 \u00b1 0.5 mm [95% CI: 6.1 – 18.9\nmm], p=0.063).<\/p>\n\n\n\n

Similarly,\nwhen assayed against K. pneumoniae<\/em>, the leaf extract mean inhibition zone diameter (24 \u00b1 0.0 mm\n[95%CI: 24 – 24 mm]) was significantly wider than the stem or root extract zone\ndiameters (17.5 \u00b1 0.5 mm [95% CI: 11.1 – 23.9 mm], p=0.024; and 17.0 \u00b1 0.0 mm\n[95% CI: 17.0 – 17.0 mm], p= cannot be estimated, respectively).<\/p>\n\n\n\n

Finally, when assayed against E. coli<\/em>, the leaf extract mean inhibition zone diameter (19.0 \u00b1 1.4 mm [95%CI: 6.3 – 31.7 mm]) tended to be wider than the stem or root extract zone diameters (15.0 \u00b1 0.0 mm [95% CI: 15.0 \u2013 15.0 mm], p=0.078; and 15.5 \u00b1 0.5 mm [95% CI: 9.1 – 21.9 mm], p=0.066, respectively).<\/p>\n\n\n\n

Table 1: E. coli<\/em> growth inhibition zone diameters from bioassays that used Commercial antibiotics. Trimethoprim-Sulfa =Trimethoprim-Sulfamethoxazole.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n
\n

 <\/strong>Commercial Antibiotic<\/strong><\/p>\n<\/td>\n

\n

Inhibition Zone Diameter (mm)<\/strong><\/p>\n<\/td>\n

\n

Interpretation based on the EUCAST Breakpoint<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Gentamicin<\/p>\n<\/td>\n

\n

22<\/p>\n<\/td>\n

\n

Sensitive<\/p>\n<\/td>\n<\/tr>\n

\n

Trimethoprim-Sulfa<\/p>\n<\/td>\n

\n

27<\/p>\n<\/td>\n

\n

Sensitive<\/p>\n<\/td>\n<\/tr>\n

\n

Amoxicillin<\/p>\n<\/td>\n

\n

13<\/p>\n<\/td>\n

\n

Resistant<\/p>\n<\/td>\n<\/tr>\n

\n

Erythromycin<\/p>\n<\/td>\n

\n

10<\/p>\n<\/td>\n

\n

Resistant<\/p>\n<\/td>\n<\/tr>\n

\n

Chloramphenicol<\/p>\n<\/td>\n

\n

19<\/p>\n<\/td>\n

\n

Resistant<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

Table 2: K. Pneumoniae<\/em> growth inhibition zone diameters from bioassays that used commercial antibiotics. Trimethoprim-Sulfa =Trimethoprim-Sulfamethoxazole.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n
\n

 <\/strong>Commercial Antibiotic<\/strong><\/p>\n<\/td>\n

\n

Inhibition Zone Diameter (mm)<\/strong><\/p>\n<\/td>\n

\n

Interpretation based on the EUCAST Breakpoint<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Gentamicin<\/p>\n<\/td>\n

\n

25<\/p>\n<\/td>\n

\n

Sensitive<\/p>\n<\/td>\n<\/tr>\n

\n

Trimethropim-Sulfa<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

Resistant<\/p>\n<\/td>\n<\/tr>\n

\n

Amoxicillin<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

Resistant<\/p>\n<\/td>\n<\/tr>\n

\n

Erythromycin<\/p>\n<\/td>\n

\n

29<\/p>\n<\/td>\n

\n

Sensitive<\/p>\n<\/td>\n<\/tr>\n

\n

Chloramphenicol<\/p>\n<\/td>\n

\n

14<\/p>\n<\/td>\n

\n

Resistant<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

Table 3: P. mirabiris<\/em> growth inhibition zone diameters from bioassays that used commercial antibiotics. Trimethoprim-Sulfa =Trimethoprim-Sulfamethoxazole.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n
\n

 <\/strong>Commercial Antibiotic<\/strong><\/p>\n<\/td>\n

\n

Inhibition Zone Diameter (mm)<\/strong><\/p>\n<\/td>\n

\n

Interpretation based on the EUCAST Breakpoint<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Gentamicin<\/p>\n<\/td>\n

\n

24<\/p>\n<\/td>\n

\n

Sensitive<\/p>\n<\/td>\n<\/tr>\n

\n

Trimethropim-Sulfa<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

Resistant<\/p>\n<\/td>\n<\/tr>\n

\n

Amoxicillin<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

Resistant<\/p>\n<\/td>\n<\/tr>\n

\n

Erythromycin<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

Resistant<\/p>\n<\/td>\n<\/tr>\n

\n

Chloramphenicol<\/p>\n<\/td>\n

\n

16<\/p>\n<\/td>\n

\n

Resistant<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

Table 4: Antibacterial activity of leaf aqueous extracts of Tithonia diversifolia<\/em> by the Kirby-Bauer disc diffusion assay<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

 <\/p>\n

 <\/strong><\/p>\n

 <\/strong><\/p>\n

 <\/strong><\/p>\n

Bacterial isolate<\/strong><\/p>\n<\/td>\n

\n

Zone of Inhibition (mm)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Concentration of leaf extract (g\/ml)<\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n

0.625<\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n

0.333<\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n

0.165<\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n

0.083<\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n

0.041<\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n

0.021<\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n

(positive control)<\/strong><\/p>\n

Gentamicin <\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n

(negative control)<\/strong><\/p>\n

Blank disk<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

E. coli<\/em><\/p>\n<\/td>\n

\n

20<\/p>\n<\/td>\n

\n

15<\/p>\n<\/td>\n

\n

11<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

16<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n<\/tr>\n

\n

18<\/p>\n<\/td>\n

\n

14<\/p>\n<\/td>\n

\n

10<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

17<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n<\/tr>\n

\n

K. pneumoniae<\/em><\/p>\n<\/td>\n

\n

24<\/p>\n<\/td>\n

\n

18<\/p>\n<\/td>\n

\n

13<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

21<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n<\/tr>\n

\n

24<\/p>\n<\/td>\n

\n

16<\/p>\n<\/td>\n

\n

12<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

19<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n<\/tr>\n

\n

P. mirabilis<\/em><\/p>\n<\/td>\n

\n

16<\/p>\n<\/td>\n

\n

13<\/p>\n<\/td>\n

\n

11<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

22<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n<\/tr>\n

\n

15<\/p>\n<\/td>\n

\n

12<\/p>\n<\/td>\n

\n

10<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

21<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

Table 5: Antibacterial activity of stem aqueous extracts of Tithonia diversifolia<\/em> by the Kirby-Bauer disc diffusion assay<\/strong>.<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

 <\/p>\n

 <\/strong><\/p>\n

 <\/strong><\/p>\n

 <\/strong><\/p>\n

Bacterial isolate<\/strong><\/p>\n<\/td>\n

\n

Zone of Inhibition (mm)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Concentration of stem extract (g\/ml)<\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n

0.625<\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n

0.333<\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n

0.165<\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n

0.083<\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n

0.041<\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n

0.021<\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n

(positive control)<\/strong><\/p>\n

Gentamicin <\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n

(negative control)<\/strong><\/p>\n

Blank disk<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

E. coli<\/em><\/p>\n<\/td>\n

\n

15<\/p>\n<\/td>\n

\n

8<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

16<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n<\/tr>\n

\n

15<\/p>\n<\/td>\n

\n

9<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

17<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n<\/tr>\n

\n

K. pneumoniae<\/em><\/p>\n<\/td>\n

\n

17<\/p>\n<\/td>\n

\n

12<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

17<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n<\/tr>\n

\n

18<\/p>\n<\/td>\n

\n

12<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

17<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n<\/tr>\n

\n

P. mirabilis<\/em><\/p>\n<\/td>\n

\n

13<\/p>\n<\/td>\n

\n

11<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

20<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n<\/tr>\n

\n

13<\/p>\n<\/td>\n

\n

10<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

21<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

Table 6: Antibacterial activity of root extracts of Tithonia diversifolia<\/em> by the Kirby-Bauer disc diffusion assay<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

 <\/p>\n

 <\/strong><\/p>\n

 <\/strong><\/p>\n

 <\/strong><\/p>\n

Bacterial isolate<\/strong><\/p>\n<\/td>\n

\n

Zone of Inhibition (mm)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Concentration of root extract (g\/ml)<\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n

0.625<\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n

0.333<\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n

0.165<\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n

0.083<\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n

0.041<\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n

0.021<\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n

(positive control)<\/strong><\/p>\n

Gentamicin <\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n

(negative control)<\/strong><\/p>\n

Blank disk<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

E. coli<\/em><\/p>\n<\/td>\n

\n

16<\/p>\n<\/td>\n

\n

12<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

15<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n<\/tr>\n

\n

15<\/p>\n<\/td>\n

\n

12<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

15<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n<\/tr>\n

\n

K. pneumoniae<\/em><\/p>\n<\/td>\n

\n

17<\/p>\n<\/td>\n

\n

11<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

21<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n<\/tr>\n

\n

17<\/p>\n<\/td>\n

\n

10<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

16<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n<\/tr>\n

\n

P. mirabilis<\/em><\/p>\n<\/td>\n

\n

12<\/p>\n<\/td>\n

\n

10<\/p>\n<\/td>\n

\n

8<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

28<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n<\/tr>\n

\n

13<\/p>\n<\/td>\n

\n

9<\/p>\n<\/td>\n

\n

8<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

28<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\"\"<\/td>\n\n

Figure 3<\/strong>: Photograph of typical<\/strong> z<\/strong>ones of inhibition <\/strong>of E. coli growth by aqueous root extracts of <\/strong>T. diversifolia<\/em><\/strong>.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 4<\/strong>: Photograph of typical<\/strong> z<\/strong>ones of inhibition <\/strong>of <\/strong>P. mirabilis <\/em><\/strong>growth by aqueous root extracts of <\/strong>T. diversifolia<\/em><\/strong>.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 5<\/strong>: Photograph of typical<\/strong> z<\/strong>ones of inhibition <\/strong>of <\/strong>P. mirabilis <\/em><\/strong>growth by aqueous stem extracts of <\/strong>T. diversifolia<\/em><\/strong>.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Minimum Inhibitory Concentration<\/strong><\/p>\n\n\n\n

Graphs 1-3\nshow the proportion of E. coli, P.\nmirabilis <\/em>and K. pneumoniae <\/em>cells\nremaining alive at different T. diversifolia<\/em> extract\nconcentrations. In general, leaf extracts exhibited greater potency on all\nstudy isolates\ncompared to stem and root extracts. <\/p>\n\n\n\n

The\nMIC50 of leaf extract was 1.58 g\/ml on E. coli <\/em>whereas\nthe MIC50 for stem and root extracts on the same organism was 7.76 g\/ml and\n16.22 g\/ml respectively. <\/p>\n\n\n\n

The\nMIC50 of leaf extract was 0.912 g\/ml on P.mi<\/em>rabilis<\/em> \nwhereas the MIC50 for stem and root extracts on the same organism was 10.12g\/ml\nand 6.76 g\/ml respectively. <\/p>\n\n\n\n

The MIC50 of leaf extract was 3.27 g\/ml on K. pneumoniae<\/em> whereas the MIC50 for stem and root extracts on the same organism was 10.23g\/ml and 10.72 g\/ml respectively.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Graph 1<\/strong>: Proportion of E. coli <\/em><\/i>cells remaining alive versus <\/strong><\/span>T. diversifolia<\/i><\/em><\/span> extract concentration.<\/span><\/strong><\/p>\n

<\/p>\n

Click here to view Graph<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Graph 2<\/strong>: Proportion of K. Pneumoniae <\/em><\/i>cells remaining alive <\/strong><\/span>
versus T. diversifolia<\/em><\/i> extract concentration<\/span><\/strong><\/p>\n

<\/p>\n

Click here to view Graph<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Graph 3<\/strong>: Proportion of Protius mirablis <\/em><\/i>cells remaining alive versus T. diversifolia<\/em><\/i> extract concentration<\/strong><\/span><\/p>\n

<\/p>\n

Click here to view Graph<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

<\/p>\n\n\n\n

Discussion<\/strong><\/p>\n\n\n\n

The\npresent study is the first to show the in vitro antimicrobial activity of aqueous extracts of Malawian T. diversifolia<\/em>\nagainst the following laboratory-adapted bacterial isolates\nfrom patients:\nE. coli<\/em>, K. pneumoniae<\/em> and P. mirabilis<\/em>. The antibacterial\neffect of T. diversifolia<\/em> leaf and\nstem extracts was similar to that of Gentamicin, a prescription drug which was\nused as a control antibiotic. Intriguingly, leaf extracts were observed to\npossess greater potency than the other plant parts. <\/p>\n\n\n\n

These findings are in broad agreement\nwith those obtained in previous studies. John-Dewole et al. (2013) observed\nthat aqueous extracts of T. diversifolia<\/em>\nproduced a 10mm zone of inhibition against E. coli20<\/sup>. Similarly, in\na study by Liasu and Ayandele (2008), it was observed that at a concentration\nof 10mg\/ml, aqueous extracts of T.\ndiversifolia<\/em> leaves exhibited a zone of inhibition of 17 mm against E.\ncoli<\/em>21<\/sup>. Compared with the potency of the afore-cited aqueous leaf\nextracts, the current study reports lesser potency for the same plant part.\nThis may be due to a difference in phytomedicine (glycosides, flavonoid,\ntannins, terpenoids, steroids, glycosides, carbohydrates, proteins and\nphenols) content that comes with geographical location of the plant. One\nlimitation of the current study is that no phytochemical screening has been done\nyet at the time of writing this paper.<\/p>\n\n\n\n

Previous phytochemical analyses of T. diversifolia<\/em> revealed the presence of glycosides, flavonoid,\ntannins, terpenoids, steroids, glycosides, carbohydrates, proteins and\nphenols in aqueous extracts22,23<\/sup>. These phytochemicals have been\nproved to harbor pharmacological effects and are among raw materials used in\nchemical synthesis of some drugs used in orthodox medicine. Tannins, for\nexample, have demonstrated antibacterial, anticancer and anti-inflammatory\neffects20, 24, 25<\/sup>. Notably, a number of phytochemical analyses reported the concentration of these bioactive compounds to be the greatest in the leaf extracts when compared to the stem or root extracts of T.\ndiversifolia<\/em>13,20,26<\/sup>. These findings of greater concentration of\nthe aforementioned known bioactive phytochemicals in leaves other than in the\nstems or roots, may explain the present study\u2019s observation of greater\nantibacterial activity\/potency in T.\ndiversifoli<\/em>a<\/em> leaf\nextracts compared to the stem or root extracts. <\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

Crude\naqueous extracts of Tithonia diversifolia<\/em> leaves showed remarkable\ngrowth inhibitory activity against E. coli, K. pneumoniae and P. mirabilis\nisolated from patients. These findings support the traditional use of Tithonia\ndiversifolia<\/em> in infectious processes due to these pathogens and further\nstrengthens recommendations for additional work to isolate and characterize the\nbioactive chemical compounds responsible for the observed antibacterial\nproperties of the plant. <\/p>\n\n\n\n

The examination\nof antibacterial activity in only aqueous extracts of Tithonia represents a\nmajor limitation of the current study. However, organic solvents were excluded\nbecause this study aimed to investigate activity in extracts that represent\ntraditional methods of herbal medicine preparation in the social settings of\nthe study. Intriguingly, in contrast to the findings in the current study, other\nauthors previously demonstrated insignificant antibacterial activity in aqueous\nleaf and root extracts of Tithonia compared to various organic solvents27<\/sup>.<\/p>\n\n\n\n

Antimicrobial resistance (AMR) poses a critical threat to global public health and modern health care systems. In 2019 a systematic paper by the Institute for Health Metrics and Evaluation revealed that at least 1.27 million deaths were linked to AMR in 2019 and that an estimated 4.95 million people who died in 2019 suffered from drug-resistant bacterial infections. It was further reported that the largest of this mortality burden occurred in the sub-Saharan Africa region28<\/sup>. This scenario represents a big challenge to the realization of sustainable development goals for the region29<\/sup>. The present study is among many that have demonstrated that medicinal plant species from Africa\u2019s rich forests represent a largely an unexploited resource for the discovery of new antibiotics to overcome the menacing scourge of AMR in the world today.<\/a><\/p>\n\n\n\n

Acknowledgement<\/strong><\/p>\n\n\n\n

Authors would like to thank Dr Baxter Kachingwe\nof Kamuzu University of Health Sciences, Malawi, for helping with sourcing some\nof the antibiotics used in this study. A word of gratitude is also extended to Mr.\nJeverson Mwale for his assistance in the Microbiology laboratory.<\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

The authors declare no conflict of interest,\nfinancial or otherwise.<\/p>\n\n\n\n

Funding Sources<\/strong><\/strong><\/p>\n\n\n\n

The Malawi University of Science\nand Technology supported the research with funds drawn from NORHED II project\non maternal and neonatal health at AMS. The project no. QZA-21\/0159.<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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     CrossRef <\/a><\/li><\/ol>\n","protected":false},"excerpt":{"rendered":"

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