Effect of Zinc Oxide Nanoparticles on Loaded Antibiotics Against Multidrug-Resistant Acinetobacter spp

Background: Metal oxide nanoparticles (NPs) have shown promising efficacy for combating bacterial resistance due to their antibacterial properties. This research investigated the effect of zinc oxide NPs (ZnO-NPs) on the antibacterial activity of conventional antibiotics including ciprofloxacin (CIP), cefotaxime (CTX), and colistin (CST) against multidrug-resistant Acinetobacter isolates. Methods: The disc diffusion method was performed to detect the pattern of antibiotic resistance in isolates. The synthesized ZnO-NPs via the solvothermal method were characterized by field emission scanning electron microscopy (FESEM), X-ray diffraction (XRD), and energy-dispersive X-ray spectroscopy (EDS). Finally, the broth microdilution technique was conducted to demonstrate the antibacterial activity of CIP, CTX, and CST antibiotics with and without a sub-inhibitory concentration of ZnO-NPs. Results: XRD, EDS, and FESEM results confirmed the crystalline structure of ZnO-NPs, and the average size was 100±58.68 nm. All isolates were discovered to be of multidrug-resistant (MDR) type and fully susceptible to CST. The antibacterial activity of CTX and CIP was restored when combined with a sub-inhibitory level of ZnO-NPs (0.25 mg/L), and the highest activity was obtained at the concentrations of 32 μg/mL CTX and 8 μg/ mL CIP. Eventually, ZnO-NPs showed a synergistic effect on the antibacterial properties of CST against MDR

doses in combination than when administered alone, and thus helping in overcoming the problems of resistance and adverse side effects (16). Accordingly, this study focused on evaluating the possible effect of ZnO-NPs to either regenerate or improve the antimicrobial activity of CIP, CTX, and CST against multidrug-resistant Acinetobacter.

Methods
All the chemicals and media were prepared from Merck (Darmstadt, Germany). The antibiogram discs were purchased from the MAST Company, UK. No human was involved in this descriptive study, and Acinetobacter isolates were acquired from discarded clinical microbiology plates collected during the diagnostic testing of Day Hospital, Tehran, Iran. The isolates were grown on brain heart infusion agar for 24-48 hours at 35°C. Their purity and identity were confirmed by macroscopic, microscopic, and standard biochemical methods.

Fabrication of ZnO Nanofluids
ZnO-NPs were synthesized using the solvothermal process according to the protocol represented by Ashtaputre et al (17). To stabilize ZnO nanofluids for antimicrobial assessments, glycerol and ammonium citrate were applied as the base fluid and dispersant, respectively. ZnO-NPs and ammonium citrate, with equal ratios, were completely mixed with glycerol solution by a magnetic stirrer at ambient temperature for 24 hours (18).

ZnO-NPs Characterization
The synthesized ZnO-NPs were characterized by several techniques. X-ray diffraction (XRD) patterns were used to determine the crystal structure of NPs. The XRD was accomplished at ambient temperature using an analytical X-Pert Pro diffractometer with Cu Kα radiation (λ = 1.54056 Å, voltage: 40 kV, current: 40 mA) and in the range of 10°-90° (2Ө) at an angular speed of 0.02°/s. Field emission scanning electron microscopy (FESEM Zeiss Sigma VP FE-SEM) on gold-coated samples was applied to identify the morphology of ZnO-NPs. Further verification was performed by energy-dispersive X-ray spectroscopy (EDS) analysis, which demonstrates the existence of Zn in the intended NPs (19).

Evaluation of the Effect of ZnO-NPs With Antibiotics (i.e., CIP, CTX, and CST) Against Multidrug-Resistant Isolates
Based on the antibiotic resistance pattern, CTX, CIP, and CST were selected for the next experiments. Then, the broth microdilution method was conducted according to the applied procedure by Balouiri et al (21) to determine the minimum inhibitory concentration (MIC). Briefly, a two-fold dilution of ZnO-NPs (0.0625-2 mg/mL), CTX (2-64 µg/mL), CIP (1-16 µg/mL), and CST (0.5-16 µg/mL) was prepared in 100 µL of Mueller-Hinton broth (MHB) in the wells of each row in a microtiter plate. Next, each well was inoculated with 50 μL of standardized microbial inoculum equal to the 0.5 McFarland turbidity. The microtiter plates were incubated at 35°C for 18-24 hours. The turbidity of all wells was determined at 630 nm using an ELSA microplate reader (Bio-Tek, Winooski, USA) every 2 hours. In addition, the growth of isolates was assayed in an ammonium citrate/glycerol mixture lacking ZnO-NPs. The antibiotic concentrations were calculated by Eq. (1) as follows: 1 .

V olume mL Concentration g mL W eight mg
Potency g mg µ µ = To assess the effect of ZnO-NPs on restoring and improving the antibacterial properties of common antibiotics, isolates were exposed to various concentrations of CAZ (16-64 µg/mL), CIP (4-16 µg/mL), and CST (0.5-2 µg/mL) combined with the sub-inhibitory concentration of ZnO-NPs (1/2 MIC, 0.25 mg/mL) for 24 hours. The growth of isolates was evaluated based on the aforementioned method. Additionally, the inoculated MHB, free of ZnO-NPs and antibiotic, was considered as a positive control. The antibacterial effectiveness was represented as the MIC and the mean of growth inhibition percentage (GI%) according to the positive control growth. GI% for each concentration was determined by Eq.

Statistical Analysis
In this study, all outcomes were expressed as mean values with their standard deviations (mean ± SD). SPSS 20 (SPSS, Chicago, IL) was used for statistical analysis. Oneway analysis of variance and Tukey's tests were applied for statistical data analyses and multiple comparisons, respectively. All tests were performed in triplicate, and the level of P < 0.05 was considered statistically significant.

Antimicrobial Resistance Pattern
Data on the antimicrobial resistance frequency of Acinetobacter isolates are presented in Table 1. All isolates showed a considerable level of resistance to the most tested antibiotics. Full resistance was determined against TIC, MEM, PIP, CTX, CIP, AM, and, AMX/CLV acid meanwhile full susceptibility was observed to CST. Moreover, all isolates represented to be resistant to different classes of antibiotics. CST, CTX, and CIP were selected for the following experiments. The EDS data analysis of the ZnO-NPs was implemented by field emission EDS. Table 2 presents the outcomes of the elemental weight percentages of the ZnO-NPs, and Figure  3 displays the EDS spectra of ZnO-NPs, including O, C, and Zn elements. These outcomes confirm the proper synthesis of the ZnO-NPs. All the identified elements by EDS are well-matched with the determined crystalline phase by XRD.

Antibacterial Activity Evaluation
The growth of Acinetobacter cells was determined with and without ZnO-NPs, CST, CTX, and CIP. Based on the results, CTX and CIP did not exert a notable inhibitory effect on growth while CST and ZnO-NPs showed considerable growth inhibitions in comparison with the control. The MIC of CST and ZnO-NPs was obtained to be 0.5-1 µg/ mL and 0.5 mg/mL, respectively, and they inhibited the cell growth in a concentration-dependent manner. Tables 3 and 4 and Figure 4 represent the mean of GI% of CTX, CIP, and CST (inhibitory and sub-inhibitory concentrations) alone and in combination with a subinhibitory concentration of ZnO-NPs (1/2MIC, 0.25 mg/ mL) against multidrug-resistant Acinetobacter cells. Based on the results, ZnO-NPs could restore the inhibitory activity of CTX and CIP and increase the antibacterial properties of CST. The highest inhibitory activity was   obtained at 8 µg/mL CIP, 32 µg/mL CTX, and 0.5 µg/mL CST.

Discussion
Acinetobacter spp. is one of the prominent MDRs among bacteria. MDR Acinetobacter is difficult to eradicate and easily spreads in the ICU (5). Therefore, new strategies should be taken into account to treat their infections. Our research focused on the possible synergistic effect of ZnO-NPs in combination with CIP and CTX to restore their antimicrobial properties against resistant Acinetobacter. Furthermore, the concomitant use of ZnO-NPs and CST was checked against multidrug-resistant isolates with the hope of reducing the functional dose of CST, thus diminishing its adverse side effects. In the current research, a high prevalence of multidrug resistance was observed among the studied Acinetobacter isolates. In other words, all isolates (100%) showed resistance to all main classes of antibiotics usually prescribed for treating infections. This finding is in compliance with the results of Noori et al (2), Ghasemi and Jalal (4), Maraki et al (23), and Vakili et al (24) demonstrating that more than 90% of the obtained Acinetobacter isolates from clinical samples were MDR. Our findings also revealed that all the isolates were completely resistant to CIP, CTX, PIP, MEM, TIC, AM, and AMX/CLV acid. Similar findings were described by Ghasemi and Jalal, Al-Naqshbandi et al, Maraki et al,and Lv et al (4,23,25,26), implying that all the studied isolates were 100% resistant to these antibiotics. Moreover, full susceptibility was observed against CST. In line with our finding, Noori et al, Maraki et al, and Rastegar-Lari et al reported a prevalence rate of 97%, 99.6%, and 100% for isolates that were sensitive to CST, respectively (2,23,27).
It was well-documented that NPs have a broadspectrum inhibitory effect against a wide range of bacterial pathogens (4,6,7,11,16). In the current research, ZnO-NPs were synthesized by the solvothermal method and well-characterized via FESEM, XRD, and EDS techniques. The antimicrobial study indicated that ZnO-NPs can be effective against the tested MDR Acinetobacter. Considering the obtained results, a direct relationship was observed between the inhibitory effect and concentration of ZnO-NPs against resistant bacteria. Some other researchers reported the dose-dependent antimicrobial manner regarding the NPs of ZnO, Ag, and Cu against Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, and Enterococcus faecalis (4,8,11). The minimum inhibitory concentration of the synthesized ZnO-NPs in this study was demonstrated as 0.5 mg/L, which is in agreement with the result of Ghasemi et al (4).
In this research, a sub-inhibitory concentration of ZnO-NPs (0.25 mg/L) in combination with all the examined     (28,29). Furthermore, ZnO-NPs considerably enhanced the growth inhibition activity of CST against MDR Acinetobacter and reduced its functional dose compared to unaided CST. In a similar study, Salman et al recommended that the combination of silver NPs and CST (Polymyxin B) resulted in increased biofilm inhibition activity against P. aeruginosa (30). Such synergistic activities between conventional antibiotics and NPs may result from inhibiting the export of antibiotics through efflux pump blockage or antibiotic entrance enhancement into the cell via bacterial membrane damage (29).

Conclusions
The combined application of ZnO-NPs with CIP and CTX separately restored their antibacterial activity considerably compared to NPs and antibiotics alone against MDR Acinetobacter. Additionally, the inhibitory dose of CST was reduced as a result of the synergistic effect of ZnO-NPs. Therefore, the use of ZnO-NPs can be a novel strategy for removing the limitations of conventional antibiotics and reviving their antimicrobial activity against resistant bacterial strains. However, this finding requires further in vitro and in vivo examination of different resistant bacteria.

Conflict of Interests
None.