We ensured that all aspects of this study complied with recognized ethical consideration. This research study has been reviewed and approved by CECOS University Institutional Review Board (IRB). Confirm that all procedures meet established ethical guidelines. Additionally, No Objection Certificate (NOC) was obtained from the Khyber Teaching Hospital (KTH), Peshawar, permitting the use of the clinical isolate of Acinetobacter baumannii solely for the purpose of this research.

This was an experimental study, specifically a laboratory based in vitro investigation conducted under controlled laboratory condition.

A clinical isolate of Acinetobacter baumannii was obtained from the Microbiology Laboratory of Khyber Teaching Hospital (KTH), Peshawar. The isolate was transported to the Institute of Integrative Biosciences (IIB) and the Medical Laboratory Technology (MLT) Laboratory at CECOS University, Peshawar, for subsequent experimental work. Nanoparticle characterization was carried out at the University of Agriculture, Peshawar. The study was conducted over a period of six (6) months.

Zinc nitrate hexahydrate (Zn(NO₃)₂·6H₂O), Manganese(II) nitrate tetra hydrate (Mn(NO₃)₂·4H₂O), Potassium hydroxide (KOH) or ammonium hydroxide (NH₄OH), Deionized (DI) water, Ethanol (for washing and dispersion), Nutrient agar and nutrient broth. Magnetic stirrer with a hot plate, Beakers (100 mL, 250 mL, 500 mL), Falcon Tubes, Glass rods, pH meter, Centrifuge machine, Analytical Balance, Ultrasonicator, Drying oven, Furnace, Photo spectrometer, Autoclave, Biosafety cabinet, Flow hood, Pipette, 96 well plate, Incubator and light incubator, Flask.

The Acinetobacter baumannii isolate, obtained from Khyber Teaching Hospital (KTH), Peshawar, was identified as multidrug-resistant (MDR), exhibiting resistance to ceftazidime (CAZ), ciprofloxacin (CIP), doxycycline (DO), imipenem (IPM), gentamicin (GEN) and amikacin (AK). The resistance profile was determined through antimicrobial sensitivity testing conducted at KTH using the Kirby-Bauer disc diffusion method. The sample was transported to the IIB Laboratory CECOS University under sterile conditions to prevent contamination.

Nutrient broth, Nutrient agar, and MacConkey agar were prepared according to the manufacturer’s instructions for the cultivation, subculturing, and isolation of bacterial isolates. The required quantity of each dehydrated medium was weighed and dissolved in the appropriate volume of distilled water with continuous stirring until completely dissolved. The pH of the media was adjusted to the recommended value (approximately 7.2 - 7.4) where necessary. The prepared media were sterilized by autoclaving at 121˚C and 15 psi for 15 - 20 minutes. After sterilization, nutrient broth was allowed to cool and was dispensed aseptically into sterile test tubes. Nutrient agar and MacConkey agar were cooled to approximately 45˚C - 50˚C before being poured into sterile Petri plates under aseptic conditions and allowed to solidify. The prepared media were stored at 4˚C until use. Nutrient broth was used for bacterial enrichment, nutrient agar for routine subculturing and maintenance of bacterial isolates, and MacConkey agar for the selective isolation and differentiation of Gram-negative bacteria based on lactose fermentation.

The known MDR Acinetobacter baumannii isolate obtained from Khyber Teaching Hospital (KTH), Peshawar, was sub cultured in the Institute of Integrative Biosciences (IIB) Laboratory, CECOS University Peshawar, to obtain fresh cultures for experimental use. The identity of the isolate was reconfirmed using standard microbiological methods, including Gram staining, catalase test and growth pattern observation on MacConkey agar, where the organism produced non-lactose fermenting colonies. Bacterial isolates were identified to the species level using the VITEK 2 automated microbial identification system (bioMérieux, France) according to the manufacturer’s instructions. Isolates identified as Acinetobacter baumannii were included for subsequent analysis. Antimicrobial susceptibility testing was performed using the Kirby-Bauer disk diffusion method on Mueller-Hinton agar. The antibiotics tested were cefepime (30 µg), amikacin (30 µg), ciprofloxacin (5 µg), meropenem (10 µg), and minocycline (30 µg). Following incubation at 37˚C for 18 - 24 h, inhibition-zone diameters were measured in millimeters and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) 2025 criteria. The isolate was used to evaluate the in vitro antibacterial activity of Mn-doped ZnO nanoparticles.

Mn-doped ZnO nanoparticles were synthesized using the co-precipitation method. Briefly, 3 g of zinc nitrate hexahydrate and 0.3 g of manganese nitrate tetrahydrate were dissolved in 50 mL of distilled water and stirred magnetically at room temperature for 10 - 15 minutes until a clear homogeneous solution was obtained. Separately, 9 g of potassium hydroxide (KOH) pellets were dissolved in 50 mL of distilled water to prepare the precipitating agent. The KOH solution was then added dropwise from a burette into the metal nitrate solution under continuous magnetic stirring at 600 - 800 rpm until a dirty brown precipitate formed, indicating complete precipitation. The resulting precipitate was collected by centrifugation at 9500 rpm for 15 minutes, washed three times with distilled water to remove residual nitrates, followed by a final wash with ethanol. After each washing step, the precipitate was resuspended and centrifuged again. The purified precipitate was dried in a hot air oven at 80˚C for 12 - 16 hours (overnight). Finally, the dried powder was calcined in a muffle furnace at 200˚C for 2 hours, 300˚C for 1 hour, and 400˚C for 1 hour using a heating rate of 5˚C/min. After cooling naturally to room temperature, the calcined product was collected as Mn-doped ZnO nanoparticles.

The synthesized manganese-doped zinc oxide nanoparticles were characterized at the Agriculture University, Peshawar, using multiple analytical techniques. X-ray Diffraction (XRD) analysis was carried out to determine the crystalline structure, phase composition, and average crystallite size of the nanoparticles. Scanning Electron Microscopy (SEM) was performed to examine the surface morphology and particle size distribution. Atomic Absorption Spectroscopy (AAS) was used to quantitatively determine the elemental composition of zinc and manganese. UV-Visible spectroscopy was employed to analyze the optical absorption properties and to estimate the band gap energy of the nanoparticles. FT-IR analysis was performed to identify the functional groups and chemical bonds associated with the synthesized Mn-doped ZnO nanoparticles. The FT-IR spectrum was recorded over the appropriate mid-infrared spectral range, and the characteristic absorption bands were analyzed and assigned based on their corresponding functional groups and previously reported ZnO-related vibrational modes.

The synthesized Mn-doped ZnO nanoparticles were accurately weighed using an analytical balance, and 10 mg of the nanoparticles was transferred into a sterile 15 mL centrifuge tube. Sterile distilled water was added to make a final volume of 10 mL, yielding a primary stock suspension with a concentration of 1 mg/mL (1000 µg/mL). The suspension was vortexed for approximately 60 seconds and subsequently sonicated in a bath sonicator for 10 - 15 minutes, with intermittent cooling intervals to prevent overheating and ensure uniform nanoparticle dispersion. Working suspensions of Mn-doped ZnO nanoparticles were prepared at concentrations of 10, 20, and 30 μg/mL. Equal volumes (100 μL) of each nanoparticle suspension and bacterial inoculum were added to the wells of a sterile 96-well microtiter plate. Consequently, the final nanoparticle concentrations in the assay wells were 10, 20, and 30 μg/mL, respectively. Accordingly, 0.20 mL, 0.40 mL, and 0.60 mL of the stock suspension were transferred into separate sterile tubes and diluted with 9.80 mL, 9.60 mL, and 9.40 mL of sterile distilled water, respectively. Each working suspension was prepared under aseptic conditions, vortexed for 30 seconds, and briefly sonicated for 2 - 5 minutes before use to minimize nanoparticle aggregation and ensure a homogeneous dispersion. All stock and working solutions were freshly prepared under sterile conditions to maintain reproducibility and prevent contamination during subsequent microbiological assays.

The antibacterial activity of Mn-doped ZnO nanoparticles against Acinetobacter baumannii was evaluated by using the broth microdilution method in sterile 96-well flat-bottom microtiter plates. Three nanoparticle concentrations (20, 40, and 60 µg/mL) were used in the assay. A fresh culture of A. baumannii was obtained by streaking the bacterial sample on MacConkey agar to observe colony morphology, followed by Gram staining to confirm the Gram-negative nature of the isolate. Although the strain was already known, these confirmatory steps were performed for record purposes. The confirmed culture was then transferred into sterile nutrient broth and incubated in a shaking incubator at 37˚C for 18 - 24 hours. The turbidity was adjusted to the 0.5 McFarland standard and diluted 1:100 in sterile nutrient broth to obtain an inoculum of approximately 1 × 10⁶ CFU/mL. 100 µL of each nanoparticle concentration was dispensed into the designated wells of the 96-well plate, followed by 100 µL of the prepared bacterial inoculum, making a final volume of 200 µL per well and a bacterial density of approximately 5 - 7.5 × 10⁵ CFU/mL. All experiments were performed in triplicate wells as technical replicates using the same bacterial inoculum. The results are presented as the mean ± standard deviation (SD) of the triplicate measurements. No inferential statistical test was performed to compare differences among the nanoparticle concentrations. As the primary objective of this study was to evaluate the antibacterial activity of Mn-doped ZnO nanoparticles against a clinical MDR Acinetobacter baumannii isolate.

To ensure the accuracy and reliability of the antibacterial assay, appropriate experimental controls were included. A sterility control (negative control) consisting of nutrient broth only, without bacterial inoculum or nanoparticles, was used to confirm that the culture medium and experimental conditions were free from contamination. A growth control (positive control) containing nutrient broth inoculated with the test bacteria but without nanoparticles was included to verify normal bacterial growth under the experimental conditions. In addition, a nanoparticle control (NP blank) comprising nutrient broth supplemented with Mn-doped ZnO nanoparticles but without bacterial inoculation was prepared to account for any background turbidity or optical interference caused by the nanoparticles during absorbance measurements. These controls ensured the validity and accurate interpretation of the antibacterial activity results.

Following inoculation, the 96-well microtiter plate was incubated at 37˚C for 18 -24 hours in a light incubator to allow bacterial growth. After incubation, bacterial growth was quantified by measuring the optical density at 600 nm (OD₆₀₀) using a microplate reader. To eliminate any interference caused by the intrinsic turbidity of the Mn-doped ZnO nanoparticles, the optical density values obtained from the nanoparticle control wells were used to correct the corresponding test wells. The antibacterial activity of the nanoparticles was then determined by calculating the percentage inhibition of bacterial growth using the corrected optical density values. This calculation enabled the quantitative assessment of the inhibitory effect of Mn-doped ZnO nanoparticles on bacterial growth.

To confirm either Mn doped zinc oxide nanoparticles was synthesized or not the Uv Vis Spectroscopy analysis was performed. A range of 300 - 600 nm was used to scan the nanoparticles. The UV-visible absorption spectra of Mn-doped zinc oxide NPs at room temperature are shown in Figure 1. A peak in the spectra at 359 nm indicated that manganese doped zinc oxide nanoparticles had formed.

Effective production of Mn-doped zinc oxide nanoparticles with a hexagonal wurtzite crystal structure was confirmed by the XRD analysis. Manganese doping does not produce any additional phases, suggesting that the material may find use in various optoelectronic and photocatalytic applications. The high degree of purity of the produced nanoparticles indicated by the lack of peaks corresponding to impurity phases. The Mn-doped zinc oxide nanoparticles’ average size was found to be 28 nm using the Scherer equation. Details were shown in Figure 2.

Figure 1. The above figure shows spectroscopy of Mn-doped ZnO NPs.

Figure 2.The above figure shows Mn-doped ZnO NPs XRD pattern.

Scanning electron microscopy (SEM) was employed to characterize the surface morphology of the synthesized manganese-doped zinc oxide (Mn-doped ZnO) nanoparticles. The SEM micrographs acquired at magnifications of ×30,000 and ×60,000 revealed irregularly shaped, agglomerated nanoparticles with non-uniform morphology. The observed aggregation is typical of metal oxide nanoparticles and is attributed to their high surface energy. The SEM analysis confirmed the successful synthesis of Mn-doped ZnO nanoparticles and provided detailed information regarding their surface morphology and structural characteristics, as shown in Figure 3.

Figure 3. SEM photographs of manganese doped zinc oxide nanoparticles.

The findings of Atomic Absorption Spectroscopy (AAS), with an emphasis on the concentrations of zinc and manganese in the generated nanoparticles, gave quantitative information on the elemental composition. This information offers a crucial foundation for understanding the overall composition and doping efficiency of the nanoparticles. The quantities of zinc (Zn) and manganese (Mn) in the synthesized manganese-doped zinc oxide nanoparticles were measured using Atomic Absorption Spectrometry (AAS). The average results are displayed below in Table 1.

Table 1. Shows the AAS result of the manganese-doped zinc oxide nanoparticles.

FT-IR spectroscopy was employed to identify the functional groups associated with the synthesized Mn-doped ZnO nanoparticles. The FT-IR spectrum exhibited a broad absorption band at 3437.8 cm⁻¹, corresponding to the O-H stretching vibration of surface hydroxyl groups and adsorbed water molecules. The absorption band at 2925.8 cm⁻¹ was attributed to C-H stretching vibrations. Peaks observed at 1595.8 cm⁻¹, 1383.7 cm⁻¹, and 1355.5 cm⁻¹ were assigned to O-H bending and residual nitrate or carbonate-related vibrations. Additional absorption bands at 1083.3 cm⁻¹, 863.4 cm⁻¹, and 770.6 cm⁻¹ were associated with metal-oxygen and lattice vibration modes. The characteristic absorption band at 575.9 cm⁻¹ was assigned to the Zn-O stretching vibration, confirming the successful formation of Mn-doped ZnO nanoparticles. Figure 4 showed the details of functional group.

Figure 4. The above figure shows FTIR analysis of the Mn-doped ZnO NPs, different peaks indicate different functional groups.

On MacConkey agar, Acinetobacter baumannii produced small, round, smooth, and translucent to slightly opaque colonies that appeared pale or colorless due to its non-lactose-fermenting nature. The absence of pink coloration distinguished it from lactose fermenters such as E. coli and Klebsiella. Growth was observed after incubation at 37˚C for 18 - 24 hours, confirming its characteristics as a Gram-negative, non-lactose-fermenting organism. As shown in Figure 5.

Figure 5. The above figure shows the colonies of Acinetobacterbaumannii.

Gram staining of the isolated colonies revealed Acinetobacter baumannii as Gram-negative coccobacillias shown in Figure 6, appearing as short, plump, pink-colored rods under the microscope. The organism was observed as non-motile and arranged singly or in pairs. The absence of purple staining confirmed its Gram-negative nature, consistent with the typical morphology of A. baumannii.

Figure 6. Gram stain of Acinetobacter baumannii showing gram negative coccobacilli.

The catalase test for Acinetobacter baumannii was performed to determine its ability to produce the enzyme catalase. Upon the addition of hydrogen peroxide to a small portion of the bacterial colony, immediate effervescence (bubble formation) was observed, indicating a positive catalase reaction. This confirmed that A. baumannii possesses the catalase enzyme, which breaks down hydrogen peroxide into water and oxygen. Details were also shown in Figure 7.

Figure 7. Catalase test of Acinetobacter baumannii showing bubble formation, indicating a positive result.

The oxidase test was performed by impregnating a sterile swab with oxidase reagent as shown in Figure 8. and then using it to pick a small portion of the bacterial colony. The swab was observed for any color change. No purple coloration developed on the swab within 30 seconds, indicating a negative oxidase reaction. This result confirmed that Acinetobacter baumannii was oxidase-negative, which is consistent with its known biochemical characteristics.

Figure 8. Oxidase test showing swab with no color change, indicating a negative result for Acinetobacter baumannii.

The antibiotic susceptibility test was performed to reconfirm the multidrug-resistant (MDR) nature of the Acinetobacter baumannii isolate, although it was previously identified as MDR. The test was carried out using the Kirby-Bauer disc diffusion method on Mueller-Hinton agar. After incubation at 37˚C for 18 - 24 hours, the isolate exhibited resistance to multiple antibiotic classes, including β-lactams, aminoglycosides, fluoroquinolones, and carbapenems. A. baumannii isolate demonstrated resistance to cefepime (30 µg), ciprofloxacin (5 µg), and meropenem (10 µg), with no measurable inhibition zones (0 mm), and to amikacin (30 µg), which produced a 10-mm inhibition zone. In contrast, minocycline (30 µg) produced an 18-mm inhibition zone and was interpreted as susceptible according to CLSI 2025 criteria. The isolate therefore demonstrated nonsusceptibility to antimicrobial agents from multiple antimicrobial classes, supporting its classification as multidrug-resistant (MDR) A. baumannii. The isolate is sensitive only to Minocycline and is therefore confirmed as multidrug-resistant (MDR) Acinetobacter baumannii. As shown in Figure 9 and Table 2.

Figure 9. Antibiotic Susceptibility test confirming Acinetobacter baumanniiisMDR.

Table 2. Antimicrobial susceptibility profile of the multidrug-resistant Acinetobacter baumannii isolate determined by the Kirby-Bauer disk diffusion method, showing antibiotic class, disk potency, inhibition-zone diameter, and susceptibility interpretation according to CLSI 2025 criteria.

The antibacterial activity of Mn-doped ZnO nanoparticles against Acinetobacter baumannii was assessed using OD₆₀₀ readings to calculate percentage inhibition and growth. The results showed a concentration-dependent inhibitory effect, where higher nanoparticle concentrations led to greater bacterial growth suppression. At 10 µg/mL, inhibition was moderate, with noticeable bacterial growth still present. Increasing the concentration to 20 µg/mL resulted in a substantial reduction in bacterial proliferation, while 30 µg/mL exhibited the highest antibacterial activity, with minimal bacterial growth observed. The growth control showed maximum OD, confirming normal bacterial growth in the absence of nanoparticles, while the sterility control showed no detectable growth, ensuring assay validity.

The percentage inhibition was calculated using the formula:

The percentage growth was calculated as: % Growth = 100 − % Inhibition. Details were also shown in Table 3. Figure 10 illustrated the effect of different concentrations of Mn-doped ZnO nanoparticles on the growth and inhibition of Acinetobacter baumannii. At 10 µg/mL, the bacterial growth was relatively higher compared to inhibition, indicating a weaker antibacterial effect at lower concentration. However, as the concentration increased to 20 µg/mL, inhibition rose notably while growth decreased, showing a concentration-dependent response. At the highest tested concentration of 30 µg/mL, inhibition reached its peak with minimal bacterial growth observed, reflecting a strong antibacterial activity of the nanoparticles. Overall, the results showed that Mn-doped ZnO nanoparticles exhibited a clear dose-dependent inhibitory effect against A. baumannii.

Table 3. OD₆₀₀ values, percentage inhibition, and percentage growth of A. baumannii at different Mn-doped ZnO nanoparticle concentrations.

Figure 10. The above bar graph shows concentration-dependent antibacterial activity of Mn-doped ZnO nanoparticles against multidrug-resistant Acinetobacter baumannii.