Antibacterial Activity and Proposed Mode of Action of Extracts from Selected Zimbabwean Medicinal Plants against Acinetobacter baumannii

Acinetobacter baumannii was identified by the WHO as a priority pathogen in which the research and development of new antibiotics is urgently needed. Plant phytochemicals have potential as sources of new antimicrobials. The objective of the study was to determine the antibacterial activity of extracts of selected Zimbabwean medicinal plants against A. baumannii and determine their possible mode of action. Extracts were prepared from the leaves of the eight plants including the bark of Erythrina abyssinica using solvents of different polarities. Antibacterial activity was evaluated using the microbroth dilution method coupled with the in vitro iodonitrotetrazolium colorimetric assay. The effect of the extracts on membrane integrity was determined by quantifying the amount of protein and nucleic acid leaked from the cells after exposure to the extracts. The effects of the extracts on biofilms were investigated. Toxicity studies were carried out using sheep erythrocytes and murine peritoneal cells. Seven out of eight evaluated plant extracts were found to have antibacterial activity. The Combretum apiculatum acetonie (CAA) extract showed the highest inhibitory activity against A. baumannii with a minimal inhibitory concentration of 125 µg/mL. The minimum inhibitory concentration (MIC) of the CAA extract caused a protein leakage of 32 µg/mL from A. baumannii. The Combretum apiculatum acetonie (CAA), C. apiculatum methanolic (CAM), Combretum zeyheri methanolic (CZM), and Erythrina abyssinica methanolic (EAM) extracts inhibited A. baumannii biofilm formation. The EAM extract was shown to disrupt mature biofilms. The potent extracts were nontoxic to sheep erythrocytes and mouse peritoneal cells. The activities shown by the extracts indicate that the plants have potential as sources of effective antibacterial and antibiofilm formation agents against A. baumannii.


Introduction
Antimicrobial resistance is one of the most serious public health threats of the twenty-frst century [1].ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) are a group of six pathogens with increasing resistance to the available antibacterial agents and virulence in humans [2].Te group is known to efectively evade the biocidal action of antimicrobial agents and is responsible for the majority of nosocomial infections [2].Te emergence and spread of multidrug-resistant (MDR) bacterial pathogens have led to increased bacterial resistance to most antibiotics used in therapy [3].In a study conducted in Zimbabwe, an increased emergence of carbapenem-resistant A. baumannii has been observed [4].
Te World Health Organization (WHO) declared carbapenem-resistant A. baumannii a priority pathogen in which studies and the development of new antibiotics are urgently needed [5]. A. baumannii is a strictly aerobic Gramnegative coccobacillus [6].Te pathogen is an opportunist that takes advantage of patients with a compromised immune system, causing a variety of infections [7].Te most common infections with A. baumannii include ventilatorassociated pneumonia, meningitis, wound and soft tissue infections, and urinary tract infections [8].Te challenges involved in the treatment of A. baumannii infections are mainly due to intrinsic and acquired resistance to multiple classes of antibiotics [9].Te mechanisms of resistance of A. baumannii include enzymatic degradation of drugs, target site modifcation, multidrug efux pumps, and membrane permeability defects [10,11].
Medicinal plants are an important source of therapeutics and a source of potential new medicines [12].Phytomedicines play a major role in the traditional system of healing, especially in developing countries [13].According to WHO, 80% of the world's population is dependent on traditional therapies [14].Plants contribute to a variety of chemical compounds called phytochemicals.Tese phytochemicals have therapeutic properties [15].Scientifc studies to determine the therapeutic potential of phytochemicals from plants are important for the development of novel antibiotics [16].Zimbabwean plants have been shown to have antibacterial, antifungal, and antiproliferative efects [17].Tese include plants from the Combretum species, Triumfetta welwitschii, Parinari curatellifolia, Vernonia adoensis, and Myricacea species [17,18].Information on the medicinal plants used in this study is shown in Table 1.Te main objective of this study was to determine the antibacterial activity of eight plant extracts from selected medicinal plants from Zimbabwe against a priority pathogen, A. baumannii.In addition, an evaluation of the possible mode of action of the most potent extracts was also carried out.31.0546E 1,483 meters above sea level), and Norton, Mashonaland Province of Zimbabwe (17.8765 °S, 30.6742E 1378.71 meters above sea level).Christopher Chapano, a taxonomist at the National Herbarium and Botanic Gardens (Harare, Zimbabwe), authenticated the plants.Te plants used in this study were Callistemon citrinus, Combretum apiculatum, Combretum zeyheri, Combretum molle, Combretum platypetalum, Erythrina abyssinica, Parinari curatellifolia, and Syzygium guineense.Te plants were collected from January 15 to January 31, 2021, during the summer period in Zimbabwe.Not all parts of the plant were used for conservation purposes.For plants used as herbal medicines in Zimbabwe, roots are used in approximately 60% of cases, followed by 35% for leaves [24].Based on their ethnomedicinal use in Zimbabwe, as well as the need for sustainable harvesting and conservation, leaves were chosen to be used in this study.

Preparation of Plant Extracts.
All the solvents used in this study were analytical grade and were obtained from Sigma-Aldrich (Darmstadt, Germany).Te leaves, bark, or fowers of the plant were dried at 40 °C in a hot air incubator (Labotec Co., Cape Town, South Africa).Te dried plant parts were ground to a fne homogeneous powder using mortar and pestle.Te cold maceration method was used to extract phytochemicals from powdered leaves, bark, or fowers.Maceration involved soaking plant materials with a suitable menstrual sample in a beaker covered with foil paper and separation of the micelle from the marc by fltration [25].In total extraction, a 20 g mass of leaf powder was placed in a beaker and 100 mL of 50 : 50 v/v dichloromethane (DCM): methanol or water: ethanol was added to the beaker to ensure that the leaf powder was submerged.Tis procedure was carried out for all the leaves of the 8 plant species including the bark of Erythrina abyssinica.
Serial exhaustive extraction involves successive extraction with solvents of increasing polarity from hexane (nonpolar) to water (polar), ensuring the extraction of a wide range of phytochemicals with diferent polarities [26].Serial extraction of a new powder sample was performed using hexane, DCM, acetone, ethyl acetate, methanol, ethanol, and water.Te container was covered with foil paper and allowed to stand at room temperature overnight with frequent agitation.Te mixture was fltered using Whatman flter paper no. 2 (Sigma Aldrich, Darmstadt, Germany).Te fltrate was allowed to dry under a fan in the fume hood cabinet.To ensure that there were no solvent residues, the samples were also dried to dry powder using a 40 °C oven for 24 hours before use.Te dried extract was weighed and stored in sterile tubes at−4 °C.In serial exhaustive extraction, the residue was retained and allowed to dry for further extraction using dichloromethane (DCM), acetone, ethyl acetate (EA), ethanol, methanol, and water.Te percentage yield for the various extracts was calculated using the following formula: Percentage yield(%) � Mass of extract obtained (g) × 100 Mass of plant powder used (g) . ( Te serial exhaustive extraction was carried out for all the leaves of the 8 plant species including the bark of Erythrina abyssinica. Cell viability after exposure to the extract was further evaluated by adding 40 μl of 0.2 mg/mL of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT) to each of the wells of the plate, and incubating the plate at 37 °C for 2 hours.Viable bacteria turn the colourless INT to red.Te lowest concentration of the extract that completely inhibited bacterial growth during overnight incubation was determined to be the minimum inhibitory concentration (MIC).In the INT assay, wells with an extract concentration that prevented colour change were considered to be the MIC.Te minimal bactericidal concentration (MBC) of the extract was determined by streaking a loopful of bacteria from wells with the ×½, ×1, and ×2 MIC concentrations on tryptic soy agar plates.Te plates were incubated overnight at 37 °C, and the MBC was determined.Te least concentration of the extract lacking visible bacterial colonies was considered to be MBC.

Efects of C. apiculatum Acetone Extract on Protein
Leakage.Te amount of proteins that leaked from A. baumannii after exposure to the Combretum apiculatum acetone (CAA) extract was determined according to the method by Du et al. [31] with some modifcations.Cells were grown overnight for 20 hours at 37 °C in a Lab-Companion incubator (S1300 shaker incubator, Jeiotech, Korea).Tey were harvested by centrifugation and suspended in 0.9% saline solution to give OD 600 1.5.Te cell suspension was exposed to the extract concentration of MIC (125 µg/mL), × ½ MIC (62.5 µg/mL), and ×2 MIC (250 µg/mL).Controls used were standard drug polymyxin B sulfate, 0.1% SDS, A. baumannii, and DMSO.Te samples were incubated at 37 °C with shaking (100 rpm) for 2 hours in a Lab-Companion incubator (S1300 shaker incubator, Jeiotech, Korea).Cell suspension aliquots of 500 μl were centrifuged at 4000 rpm for 4 minutes.To 50 μl of the supernatant, 950 μl of Bradford reagent was added to determine the protein content using the Bradford method.Bovine serum albumin (BSA) was used as a standard protein in the Bradford assay.Te amount of protein leaked from the cells was quantifed by measuring absorbance at 590 nm reading using the microplate reader (Tecan GENios-Pro microplate reader, Grödig, Austria).

Te Efects of C. apiculatum Acetone Extract on Nucleic
Acid Leakage.Te potential of the CAA extract to cause nucleic acid leakage from A. baumannii was determined using propidium iodide as described by El-Nakeeb et al. [32] with some modifcations.Propidium iodide is a red fuorescent nucleic acid stain that intercalates with the DNA of dead cells, as it penetrates only cells with disrupted membranes and is generally excluded from viable cells [33].Cells grown overnight were centrifuged and suspended in 0.9% saline solution (OD 600 1.5).Te suspension of A. baumannii cells was exposed to extract concentration of ×½ MIC (62.5 µg/mL) MIC (125 µg/mL) and ×2 MIC 2 (250 µg/mL).Untreated cells, 0.1% SDS, 3%DMSO, and polymyxin B sulfate were used as controls.Samples were incubated at 37 °C with shaking (100 rpm) for 30 minutes in a Lab-4 Advances in Pharmacological and Pharmaceutical Sciences Companion incubator (S1300 shaker incubator, Jeiotech, Korea).For each 1 mL sample, the aliquots were centrifuged at 1100 rpm for 1 minute in a microcentrifuge (Eppendorf ™ 5415 C).Te pellet was washed with 1 mL 0.9% saline solution and suspended in 3 mL of saline.A volume of 3 µL propidium iodide was added to each sample, and the solution was mixed.Te samples were kept in the dark for 10 minutes after which fuorescence was measured at excitation and emission wavelengths of 544 and 612 nm, respectively, using an f max microplate spectrofuorometer (Molecular Devices, Sunnyvale, USA).

Te Efect of the Extracts on Bioflms.
Te antibioflm activities of the extracts of CAA, CAM, CZM, and EAM were determined by staining the bioflms using crystal violet according to O'Toole [34].

Te Efect of Extracts on Bioflm Formation.
A. baumannii cells grown overnight were centrifuged at 3500 rpm for 5 minutes (Hettich Rotofx 32 centrifuge (Tuttlingen, Germany).Te remaining pellet was washed with PBS (0.5 M, pH 7.2).Cells were standardised using the 0.5 McFarland's standard to a concentration of 2 × 10 6 CFU/ ml.A volume of 1 ml of cells was added to each of the wells of a 24-well microplate.Te cells were allowed to adhere to the plate by incubating them in a shaking incubator at 37 °C for 2 hours.Stock concentrations of the extracts were prepared.An equal volume of extracts (1000 µg/mL) was added to the wells of the plate.Te 24-well microplate was incubated for 72 hours at 37 °C on a LAB Doctor Mini Incubator (MID SCI, USA).After incubation, nonadherent cells were removed by gently washing 3 times with PBS.Te plates were dried by inverting them on absorbent paper for 15 minutes.Te samples were fxed at 60 °C for an hour in a drying oven (Mermmet Universal oven, southern Germany).Te bioflms were stained with crystal violet.Te plate was gently washed with sterile water and left to dry overnight.A volume of 2.5 mL ethanol was added to each well, and 200 µL of the plate contents were transferred to a 96-well microplate.Optical density readings were taken at 590 nm.Te percentage bioflm growth was calculated using the following formula:

Microscopic Analyses of A. baumannii Static Bioflms.
Microscopic analysis of the efects of CAA, CAM, CZM, and EAM on A. baumannii bioflm structure was carried out by staining the bioflms with a crystal violet stain and visualising these under a digital microscope.An overnight culture of A. baumannii was standardised to 2 × 10 6 CFU/mL using a 0.5 McFarland standard.Te extracts (1000 μg/mL) and cells were dispensed in a ratio of 10 mL: 10 mL in Petri plates containing a sterile microscopic glass slide.Petri plates were incubated at 37 °C for 72 hours.A plate with Muller-Hinton broth was included as a control for sterility.A plate with cells not exposed to the extract served as a positive control.After the incubation period, 2.7 ml of 1.5% SDS in PBS (v/v) was added to the plates.Te plates were incubated for another 30 minutes at 37 °C.Microscopic slides were removed from the Petri plates aseptically and washed with PBS.Te bacterial bioflms were fxed to the slides using 2% sodium acetate and stained with 0.1% crystal violet.Te slides were washed and air-dried.After drying, the bioflms were visualized under a Celestron digital microscope (Celestron, California, USA) at ×40 magnifcation.

Evaluation of the Bioflm Disruption Potential of Potent
Extracts on Mature A. baumannii Bioflms.A 24-well plate was prepared as described in the static bioflm formation assay without the addition of the test sample (extracts).Te plate was incubated for 72 hours at 37 °C on a LAB Doctor Mini Incubator (MID SCI, USA).After incubation, cells were washed and the test sample (1000 µg/mL) was dispensed into the wells.Te plate was further incubated for 24 hours, and the bioflms were quantifed following the procedure for static bioflms.

Microscopic Analysis of the Efect of Potent Extracts on
Mature A. baumannii Bioflms.Microscopic analysis of the efects of the extracts of CAA, CAM, CZM, and EAM on mature bioflm structure was carried out as described in the microscopic analysis of the static bioflm formation assay without the addition of the extracts.Petri plates were incubated at 37 °C for 72 hours.After the incubation period, the microscopic slides were washed gently using PBS to remove nonadherent cells.A volume of 10 mL of the test sample (1000 µg/mL) was distributed into the Petri plates containing the microscopic slides.Te Petri dishes were incubated for 24 hours.After further incubation, the slides were prepared according to the microscopic analysis of the static bioflm formation assay procedure and visualized under a Celestron digital microscope (Celestron, California, USA) at ×40 magnifcation.

Evaluation of the Toxicities of Potent Extracts.
In vitro toxicity evaluations of CAA, CAM, CZM, and EAM extracts were investigated using the haemolysis assay and toxicity test on mouse peritoneal cells.

Toxicity Determination Using Haemolysis Assay.
Te haemolysis assay was performed as described by Malagoli [35].Blood (20 mL) was aseptically collected from an adult sheep in the animal house (the University of Zimbabwe).An equal volume of Elsevier's solution, an anticoagulant, was immediately added.Blood was centrifuged (Hettich Rotofx 32 centrifuge, Tuttlingen, Germany) at 3000 rpm for 10 minutes, and the supernatant was discarded.Te remaining residue was washed three times with PBS.Te washed cells were diluted four times with PBS.Cells (500 µL) were incubated with an equal volume of varying concentrations of extract dissolved in PBS for Advances in Pharmacological and Pharmaceutical Sciences 90 minutes at 37 °C.After incubation, the tubes were spun in a microcentrifuge (Eppendorf ™ 5415 C) at 3000 rpm for 1 min.Te resulting supernatant (200 µL) was added to 3 mL of Drabkin's reagent.An uncentrifuged mixture of erythrocyte suspension and PBS was a positive control.Te centrifuged erythrocyte suspension and bufer (PBS) were the negative control.A volume of 200 µL of supernatant aliquots was placed in Drabkin's reagent on 96-well microplates (Figure 1).Absorbance readings of the samples taken using a Tecan GENios-Pro microplate reader (Grödig, Austria) at 590 nm were used to estimate the amount of haemoglobin released.Te percentage of haemolysis for each sample was calculated using the following formula: % haemolysis � sample's absorbance − negative control absorbance positive control absorbace − negative control absorbance × 100. (4) 2.9.2.Toxicity Tests in Mouse Peritoneal Cells.Male mice (BALB c) of 20-25 g weight were collected from the animal house (the University of Zimbabwe).Te starch solution (20%) was injected intraperitoneally into 2 mice to increase the production of peritoneal cells within the mice.Te mice were left for 48 hours in plastic cages with an unlimited pelleted basal diet and water.Te peritoneal cells were isolated according to a method described by Ray and Dittle, [36].Te mice were sacrifced using the cervical dislocation method.Each mouse was then sprayed with 70% ethanol and mounted on a Styrofoam board on its back using pins.Te outer skin of the peritoneum was gently cut and pulled using scissors and forceps to expose the inner peritoneal cavity.A volume of 5 mL cold FBS (3%) and a solution of PBS were injected into the peritoneal cavity without damaging any organs.Te peritoneum was gently massaged with fngers to allow the attached cells to enter the bufer solution.A pasture pipette was used to collect as much fuid as possible.Te fuid collected was kept on ice.Cells harvested were centrifuged in a Hettich Rotofx 32 centrifuge for 10 min at 1500 rpm [37].Te cell pellet was collected and suspended in RPMI supplemented with 10% FBS and 1% PNS (penicillin, neomycin, and streptomycin).Cells were incubated overnight at 37 °C in a CO 2 , series II water-jacket incubator (Termo Forma, Ohio, USA).Mouse peritoneal cell viability was determined using a trypan blue exclusion assay.Cells were stained with 0.4% trypan blue and manually counted using a haemocytometer beneath a Celestron digital light microscope (Celestron, California, USA) using a ×10 objective lens.Te percentage of cell viability was 98%.
Te MTT assay described by Kundishora et al. [38] was used to determine toxicity.Te extracts of CAA, CAM, CZM, and EAM were prepared to give concentrations of 1.25, 2.5, 5, 10, and 20 mg/ml.Each well contained 100 μL of test extract and 100 μL of 0.5 × 10 5 cells/mL in supplemented RPMI.Cells exposed to the standard anticancer drug doxorubicin (10 µg/mL) were used as a positive control.Cells in RPMI were used as negative control.Cells were incubated in 96-well plates (Figure 2) in the presence of extracts for 24 h at 37 °C in a CO 2 , water-jacketed incubator series II (Termo Forma, Ohio, USA).

Statistical Analysis.
Graph Pad prism for windows (Graph Pad Software Inc., San Diego, California, USA) version 8.0.1 was used to analyse data from the results obtained in this study.Te one-way analysis of variance test (ANOVA) with Dunnett's multiple comparison test was used to determine the level of signifcance, where all treated samples were compared to the control.Statistical signifcance was considered if P values < 0.05.

Antibacterial Activities of Extracts.
Extracts from 8 medicinal plants from Zimbabwe prepared from solvents of varying polarities were screened for antibacterial activity against A. baumannii.Polymyxin B sulfate was used as a positive control.Te standard drug had a MIC of 2 µg/mL.A total of 7 out of 8 medicinal plants evaluated for antibacterial activity were found to have an antibacterial efect against A. baumannii.Te leaf extracts that showed the highest activity against A. baumannii for each plant are shown in Table 2. Te CAA extract was the most potent with an MIC of 125 µg/mL (Figure 3).Other potent extracts included the CAM extract with a MIC of 250 µg/mL, EAM extract (MIC-500 µg/mL), S. guineense ethyl (SGE) acetate extract (MIC-500 µg/mL), and CZM extract (MIC-1000 µg/ mL).

Te Efects of C. apiculatum Acetone Extract on the
Membrane Integrity of A. baumannii.Te ability and extent of the CAA extract to cause membrane damage to A. baumannii as a mode of action were assessed using the protein leakage and nucleic acid assay.In the protein leakage assay, protein content was estimated using the bovine serum albumin (BSA) standard curve after A. baumannii were exposed to varying concentrations of the extract, as shown in Figure 4. Te extract managed to cause leakage of protein from the concentrations of ½ MIC, MIC, and ×2 MIC.Te protein concentration leaked was 24, 32, and 41 µg/mL for ½ MIC, MIC, and ×2 MIC concentrations, respectively.

6
Advances in Pharmacological and Pharmaceutical Sciences In the nucleic acid assay, the fuorescence of propidium iodide of the diferent samples exposed to the CAA extract is shown in Figure 5. Acetone extract did not cause signifcant leakage of nucleic acids from A. baumannii compared to unexposed cells, as the fuorescence of propidium iodide was comparable to that of the control.An increased fuorescence of propidium iodide was observed in cells treated with polymyxin B and 0.1% sodium dodecyl sulfate.Tere was a signifcant diference in the bioflm formed when cells were exposed to the extracts compared to the bioflm formed in the unexposed cells.At 1000 μg/mL, the extracts of CAA, CAM, CZM, and EAM resulted in only 42%, 50%, 42%, and 41% bioflm formation by A. baumannii, respectively (Figure 6).

Microscopic Analyses of Static A. baumannii Bioflms.
Microscopic analysis of the efects of CAA, CAM, CZM and EAM extracts on the formation of A. baumannii bioflms was carried out by staining the bioflms using a crystal violet stain and viewing the microscopic sides at ×40 magnifcation Advances in Pharmacological and Pharmaceutical Sciences Advances in Pharmacological and Pharmaceutical Sciences using a Celestron ™ digital microscope (Celestron, LLC, California, USA). A. baumannii cells exposed to standard drug or extracts (CAA, CZM, and EAM) showed dispersed planktonic cells.Cells exposed to the CAM extract showed a partially disrupted bioflm structure.Unexposed cells of A. baumannii showed an undisturbed bioflm structure (Figure 7).

Evaluation of the Bioflm Disruption Potential of Extracts
in Mature Bioflms.Te potential of the extracts to disrupt mature bioflms was evaluated using the crystal violet assay.Te CAA, CAM, and CZM extracts did not show antibioflm activity on mature bioflms.Exposure of mature bioflms to the EAM extract resulted in reduction of bioflms.Only 57% bioflm growth was observed (Figure 8).

Microscopic Analysis of Mature Bioflms.
Te potential disruption of mature bioflms by the CAA and CAM, CZM, and EAM extracts in mature bioflms of A. baumannii was determined.Te efects were observed on the slides under a Celestron TM digital microscope (California, USA) at a ×40 magnifcation.When mature bioflms were exposed to the EAM extract, a partial bioflm disruption was observed.Te mature bioflms of A. baumannii treated with CAA, CAM, and EAM extracts showed a compact bioflm structure.Bioflm structures were not observed in bioflms exposed to polymyxin B (Figure 9).

In Vitro Toxicity: Haemolysis Assay.
Te efect of the extracts of CAA, CAM, CZM, and EAM on sheep erythrocyte membrane integrity was investigated using the Drabkin cynamethemoglobin method.Te activity of the four extracts is expressed as a percentage haemolysis.Te efect of the extracts on sheep erythrocytes is shown in Figure 10.Tere was an increase in haemolysis activity as the extract concentration increased.Te intensity of the colour increased with increasing release of haemoglobin.Plant extracts are deemed toxic to red blood cells if the degree of haemolysis is greater than 30% (ISO 100993 −5, 2009).CAA, CAM, and EAM extracts were considered nontoxic since they showed a percentage haemolysis of 30%, 23%, and 26.4% at 20 mg/mL, respectively.Te CZM extract showed low toxicity with a percentage haemolysis of 33% at 20 mg/ mL, as seen in Figure 11.Advances in Pharmacological and Pharmaceutical Sciences

Efect of CAA Extract on Mouse Peritoneal Cells.
Te cytotoxicity efect of four extracts that showed antibacterial activity against A. baumannii was tested on immune cells elicited from the peritoneal cavities of mice.Te efect was determined using the MTT assay.Te yellow tetrazolium MTT salt was reduced by dehydrogenase enzymes in metabolically active cells, giving a purple colour [39].Te intensity of the purple colour was used to calorimetrically measure viable cells.As can be seen in Figure 12, the four extracts were not toxic to immune cells.Exposure to the extract resulted in an increase in cell density with an increase in extract concentration.All extracts signifcantly increased peritoneal cell viability with the CZM extract giving the most increase of 6 fold (Figure 13).Te media+extract-only wells were used to subtract the efect of coloured extracts.

Discussion
Te increase in microbial resistance to conventional antibiotics has raised serious concerns in the treatment of infectious diseases [40]. A. baumannii is one of the serious multidrug-resistant pathogens.Te need to fnd new Advances in Pharmacological and Pharmaceutical Sciences potential antibacterial compounds against MDR A. baumannii is urgent [41].Plants are a valuable source of new bioactive compounds with antimicrobial activities for pharmaceutical development [42].Tey are efective in the treatment of infectious diseases, while at the same time alleviating many of the side efects that are often associated with synthetic antimicrobial agents [43].
Te study aimed to evaluate the antibacterial activities of extracts of eight medicinal plants in Zimbabwe against a frst-priority pathogen of the WHO A. baumannii.A total of seven out of eight medicinal plants evaluated for antibacterial activity were found to have an antibacterial efect against A. baumannii.Te serial exhaustive extraction gave the best result compared to extractions with DCM-methanol or water-ethanol solvents.Te CAA extract was the most potent inhibitor of the growth of A. baumannii.Other extracts that were more potent included CAM, EAM, SGE, and CZM extracts.Te crude extracts that were potent inhibitors of growth were extracted using polar solvents.Polar solvents are known to extract alkaloids, favonoids, terpenoids, saponins, and tannins [44], which may be attributed to the antibacterial activity observed in this study.Te choice of solvent and the sequence of extraction in natural products preparation depend on the specifc compounds you aim to extract and their solubility properties.Generally, extraction can be done in a sequence from Advances in Pharmacological and Pharmaceutical Sciences nonpolar to polar or polar to nonpolar, depending on the desired outcomes [25].Based on our results, we think it is best to have the 8 extracts in a sequential order ranging from nonpolar to polar as it will be easier to remove the organic solvents because of their low boiling points under reduced pressure.Tere is a need for improvement of the solvent ratio so as to optimize the ratio solvent to plant materials to ensure complete extraction of target compounds and to make sure that each extraction step is performed thoroughly before moving to the next solvent [45].Tere could be a need for ultrasonication to enhance solvent penetration as this may improve extraction efciency.In a study by Mangoyi et al. [20], Combretum species including C. apiculatum and C. zeyheri were found to possess inhibitory activity against C. albicans and C. krusei.When phytochemical identifcation was carried out, favonoids were found to have the most antifungal activity.In other studies, the C. apiculatum acetone extract exhibited anti-infammatory and anthelmintic activity [46].In another study, volatile oils from cinnamon, clove, and tree basil were found to possess antibacterial activity against MDR A. baumannii with MBC of 0.5, 1, and 2 mg/mL [47].Te study used an increased concentration of extracts compared to our study where our highest concentration was 1000 µg/ mL.Studies by Miyasaki et al. [48] demonstrated that a pure compound norwoginin, a favone extracted from Scutellaria baicalensis, had signifcant antibacterial activities against A. baumannii with a MIC of 126 µg/mL.According to the literature, the common type of compounds in C. molle, C. apiculatum, C. zeyheri, and S. guineense are favonoids particularly quercetin and kaempferol.In addition, tannic acid and ellagitanins are also found in all the 4 species [49].In our own studies, we were able to isolate β-sitosterol from both P. curatellifolia and C. platypetalum [50,51].Te chemical components of the extracts were not compared in this study as it was initial screening against this number 1 priority pathogen to determine which plant species would produce antibacterial bioactive components.
Secondary metabolites from plants can afect microbial cells by altering and permeabilizing the membrane structure [52].Te ability and extent of CAA to cause membrane damage of A. baumannii as a mechanism of action using the protein and nucleic acid leakage assay.Exposure of cells to × ½ MIC, ×1 MIC, and ×2 concentrations of the CAA extract resulted in an increased leakage of proteins indicating membrane disruption.In the nucleic acid assay, the acetone extract did not cause signifcant leakage of nucleic acids from A. baumannii compared to untreated cells.In a similar study, Hao et al. [53] demonstrated that Litsea cubeba essential oil can disrupt the integrity of the A. baumannii membrane and contribute to the leakage of nucleic acids and proteins.Leakage of cell components can lead to inhibition of bacterial growth [54].Other studies have reported the elimination of reactive oxygen species and protein leakage as A. baumannii, a leading nosocomial pathogen, can persist in hospital environments and medical devices due to its ability to form bioflms [56].Bioflms can be defned as a complex community of microbes that can be found attached to a surface or form aggregates [57].formation of these bioflms is one of the leading causes of multidrug resistance in bacteria and ultimately increases the cost of treating patients [58].Te integrity of cells in the bioflm matrix limits the permeability of antibiotics [55].In the study, the inhibitory activity of the bioflm formation and the disruption potential of mature bioflms of CAA, CAM, CZM, and EAM extracts against A. baumannii were investigated.All four extracts inhibited the formation of bioflms in A. baumannii.Tis work is in contract with our previous study [59] in which we showed that the extracts and a pure compound of tormentic acid, isolated from Callistemon viminalis, had no efect on A. baumannii.Tere was a signifcant diference in the bioflm formed when cells were exposed to the extracts compared to the bioflm formed in the unexposed cells.Bioflms are formed in four stages which consist of an attachment of bacteria to the surface, microcolony formation, maturation of bioflms, and dispersal of bacteria to fnd new niches [60,61].When observed under a microscope, A. baumannii cells exposed to the extracts could be seen as planktonic cells compared to the untreated cells that showed a compact bioflm structure.Terefore, the extracts inhibited the formation and maturation of microcolony in the bioflms.Other studies have indicated that the most efective treatment for bioflm-based infections is to inhibit the initial binding phase [58].Terefore, inhibition of the initial bioflm formation by plant extracts may provide a strategy for the prevention and treatment of bioflm-based infections caused by A. baumannii.
When the disruption potential of the extracts was investigated in mature bioflms, only the EAM extract showed activity in mature bioflms.Polar solvents such as methanol have been shown to extract the highest amount of bioactive compounds [62].Te antibioflm properties shown by the EAM extract may be attributed to the alkaloids, tannins, favonoids, and saponins found in the plant [23].Similarly, in a study by Sanchez et al. [63], methanolic extracts of Opuntia fcus-indica caused a reduction in bioflm formation against A. baumannii bioflms.Phytochemical analysis revealed the presence of favonoids, coumarins, and tannins.In other studies by Karunanidhi et al. [64], Alium stipitatum hexane and DCM extracts were found to have antibioflm activity against A. baumannii and methicillin-resistant S. aureus.Te antibioflm activity of natural products is mainly based on suppression of cell adhesion and attachment, inhibition of polymer matrix formation, and decreasing virulence factor production, thus blocking the quorum sensing network and bioflm development [55,65].Te extracts might have exerted their antibioflm activity using one of these mechanisms of action.

Advances in Pharmacological and Pharmaceutical Sciences
Although plant extracts can be active against microorganisms [66], they can be toxic at high doses [67].It is necessary conduct a toxicity study to ensure the safety of the plants in animal models [68].Te haemolytic activity of any compound is an indicator of general cytotoxicity to normal healthy cells.Haemolysis is characterised by the release of haemoglobin due to lysis of the lipid bilayer membrane [69].Te efect of CAA, CAM, CZM, and EAM extracts sheep erythrocyte membrane integrity was investigated using the Drabkin cynamethemoglobin method.Haemoglobin from haemolysed cells is oxidized to methemoglobin, then cynamethemoglobin gives a red colour that absorbs at 590 nm [70].Te intensity of the colour is proportional to the haemoglobin released.In this study, the intensity of the colour increased with increasing extract concentration.According to the International Organization for Standardization, plant extracts are considered toxic to red blood cells if the percent haemolysis is greater than 30% as indicated by ISO100993−5 [71].Tree of the extracts tested were deemed nontoxic since the percentage of haemolysis was 30% and below.Te CZM extract had a haemolytic activity of 33% and was considered slightly toxic to sheep erythrocytes.Te three extracts CAA, CAM, and EAM were considered suitable for the preparation of herbal cream, since they were nontoxic at higher concentrations of 20 mg/mL.
Macrophages are immune cells that participate in both innate and adaptive immunity.Peritoneal cavity macrophages are commonly used in in vitro assays, as they are more stable in their functionality and phenotype [72].Te presence of a large number of naïve macrophages makes the peritoneal cavity a preferred site for the collection of macrophages [36,73].Te potential of plant extracts to inhibit murine macrophage growth was used as an indication of toxicity.Te cytotoxicity efect of the extracts of CAA, CAM, CZM, and EAM was evaluated using the MTT assay.Te four extracts were nontoxic to mouse peritoneal cells but instead promoted the growth and survival of these cells with increasing extract concentration compared to the untreated cells.Several studies have also shown that plant extracts are nontoxic but have an immune-stimulatory efect on murine macrophages.Adegbaju et al. [72] showed that plant extracts from Celosia argentea were not toxic to RAW264 macrophages and the Celosia agentea acetone extract showed increased cell proliferation.Mapfunde et al.
[74] also had similar fndings.In that study, they found that C. zeyheri alkaloid, saponin, and ethanol extracts were nontoxic to mouse peritoneal cells and Jurkat T-cells.Plants can be immunomodulators that can directly inhibit or stimulate the immune system [75].Modulation of the immune system involves infuencing cytokine production, lymphocyte proliferation, and macrophage stimulation [76].Te increase in cell proliferation shown by the extracts suggests that the extracts can stimulate an immune response which is essential in the fght against bacterial infections.

Conclusions
Te extracts of CAA, CAM, CZM, and EAM showed the most signifcant antibacterial activity against A. baumannii.
Te CAA extract has shown to damage bacterial cell membranes, resulting in protein leakage.All four extracts inhibited the formation of bioflms of A. baumannii.Te EAM extract disrupted the mature bioflm structure of A. baumannii.Te presence of antibacterial and antibioflm formation activities shown by the extracts indicates that the plants have potential as sources of efective antibacterial and antibioflm formation agents against bacterial infections caused by A. baumannii.Toxicity studies showed that the 4 extracts are nontoxic, so further studies can be carried out in animal models without toxicity to normal cells.Te study was limited to extracts and in vitro studies, so further studies can be carried out to determine the active compounds responsible for the antibacterial and antibioflm activities.In vivo studies of antibacterial activity in rodent models are also necessary to fully investigate the antibacterial mechanism of action of the four extracts.

Figure 1 :Figure 2 :
Figure 1: Haemolysis template plate for the efects of the extracts on sheep erythrocytes.Illustrative representation of the plate layout set to determine the haemolysis of sheep erythrocytes exposed to extracts of C. apiculatum acetone (CAA), C. apiculatum methanol (CAM), C. zeyheri methanol (CZM), and E. abyssinica methanol (EAM).All extracts ranged from 1.25, 2.5, 5, 10, and 20 mg/mL.Te negative control contained centrifuged cells in Drabkin's reagent.A positive control with 100% haemolysis was obtained by mixing uncentrifuged erythrocytes with Drabkin's reagent.

>Figure 3 :Figure 5 : 1 %Figure 4 :
Figure 3: Te efects of the most potent extracts of C. apiculatum on the growth of A. baumannii: (a) are the data analysed and (b) is a picture of the efects of the acetone and DCM extracts.Extract concentrations ranged from 0 to 1000 μg/mL.Bacteria used were 2 × 10 6 CFU/mL.Values are expressed as mean cell density at a wavelength of 590 nm wavelength ± the standard deviation (n � 4).Te two extracts of C. apiculatum had a signifcant efect on the growth of A. baumannii.

Figure 6 :Figure 7 :
Figure 6: Qualitative analysis of the efects of extracts and polymyxin B sulfate on bioflm formation in A. baumannii.(a) Te negative control contained only broth.Te error bars indicated the standard deviation from the mean (n � 4).Te asterisks * indicate statistically signifcant diferences compared to the positive control (unexposed cells), which represented cells without any extract, where * * * * denotes P < 0.0001.At 1000 μg/mL, extracts from CAA, CAM, CZM, and EAM resulted in only 42%, 50%, 42% and 41% bioflm formation by A. baumannii, respectively.Slide (b) shows a picture of the 24-well plate used in the assay.

Figure 8 :Figure 9 :
Figure 8: Qualitative analysis of the efects of extracts and polymyxin B sulfate on the formation of mature bioflms in A. baumannii.(a) Te negative control contained only broth.Te error bars indicated the standard deviation from the mean (n � 4).Te asterisks () indicate statistically signifcant diferences compared to the positive control (unexposed A. baumannii), which represented cells without any extract, where * * * * denotes P < 0.0001.Exposure of cells to CAA, CAM, and CZM extracts did not show antibioflm activity on mature bioflms.Exposure to the EAM extract resulted in the reduction of mature bioflms.Slide (b) shows a picture of the 24-well plate used in the assay.

Figure 10 :Figure 11 :
Figure 10: Te efects of exposing sheep erythrocytes to the extracts.A 96-well microplate was used to determine the haemolysis activity of sheep erythrocytes exposed to extracts of C. apiculatum acetone (CAA), apiculatum methanol (CAM), C. zeyheri methanol (CZM), and E. abyssinica methanol (EAM).Te negative control contained centrifuged cells in Drabkin's reagent.A positive control with 100% haemolysis was obtained by mixing uncentrifuged erythrocytes with Drabkin's reagent.Te intensity of the colour increased with an increase in the extract concentration of 1.25, 2.5, 5, 10, and 20 mg/ml.

Figure 12 :
Figure 12: Te efect of the extracts on mouse peritoneal cells.An image of a 96-well plate used for the MTT assay for (a) C. apiculatum extracts of acetone (CAA) and methanol (CAM) and (b) extracts of C. zeyheri methanol (CZM) and E. abyssinica methanol (EAM).Controls were cells only (negative control) and doxorubicin (positive control).Te intensity of the colour increased with increasing extract concentration.Only wells from the media + extract were used to subtract the efect of coloured extracts.

Figure 13 :
Figure 13: Quantitative determination of the efects of exposing mouse peritoneal cells to the extracts.Te cytotoxic efect of the C. apiculatum acetone (CAA), C. apiculatum methanol (CAM), C. zeyheri methanol (CZM), and E. abyssinica methanol (EAM) extracts in mouse peritoneal cells.Cell density increased with increasing extract concentration.Te values are for mean ± standard deviation (error bar) for n � 4. Te asterisks indicate a signifcant diference from the control * * * * P < 0.0001.Tere was ×3.4,×3, ×6, and ×3.6 fold increase in cells as a result of exposure to CAA, CAM, CZM, and EAM extracts, respectively.
2.1.Plant Collection and Authentication.Te medicinal plants used in this study were collected from the Centenary communal lands (geographical coordinates 16.7294 °S, 31.1166°E; 1213.42 meters above sea level) in the Mashonaland Central Province of Zimbabwe, the University of Zimbabwe, Mt.Pleasant Harare Province of Zimbabwe (17.7824 °S,

Table 1 :
Zimbabwean medicinal plants evaluated for their antibacterial activity against A. baumannii in this study.

Table 2 :
Antibacterial activities against A. baumannii of leaf extracts from Zimbabwean medicinal plants.