Acinetobacter baumannii is a notorious opportunistic pathogen, frequently responsible for infections in hospitalized patients, particularly in intensive care units (ICUs) (1). Although it generally exhibits low virulence, A. baumannii can cause severe infections, often through contaminated respiratory equipment and catheters (2, 3). These infections include skin and soft tissue infections, wound infections, urinary tract infections, secondary meningitis, and bloodstream infections such as bacteremia (4). Among these, ventilator-associated pneumonia and bacteremia are particularly common, with ventilator-associated pneumonia mortality rates ranging from 40% to 70% (5). Bloodstream infections have mortality rates between 28% and 43%, while urinary tract infections, typically related to prolonged catheter use, are more common in general hospital wards (6). Wound infections are often observed in patients with severe burns or deep combat-related injuries (7). High-risk groups include the elderly, immunocompromised individuals, and those exposed to long hospital stays, colonized patients, or broad-spectrum antibacterial treatments (8, 9).

A major challenge in managing A. baumannii infections is the emergence of multidrug-resistant (MDR) strains (10). These strains exhibit resistance to multiple antibiotic classes, including beta-lactams, aminoglycosides, and fluoroquinolones, largely due to genes located on mobile genetic elements such as transposons and integrons (11). Carbapenem antibiotics like imipenem and meropenem are commonly used to treat A. baumannii infections, but resistance to these drugs ranges from 28% to 85% (12).

Fluoroquinolones, which inhibit bacterial DNA replication by targeting DNA gyrase and topoisomerase IV, are key in treating A. baumannii infections (13). However, resistance to fluoroquinolones is primarily caused by chromosomal mutations in the quinolone resistance-determining regions (QRDR) of the gyrA and parC genes, leading to alterations in the enzymes responsible for DNA replication (14). The most commonly reported mutations associated with fluoroquinolone resistance occur at Ser-83 in the gyrA gene and Ser-80 in the parC gene. In contrast, genetic alterations at the gyrB (Ser83Leu) and parE (Ser80Leu) loci are not implicated in fluoroquinolone resistance (15). Additionally, plasmid-mediated quinolone resistance (PMQR) genes provide an additional mechanism of resistance, often complementing chromosomally encoded mutations (16). PMQR mechanisms include qnr peptides that protect topoisomerases and efflux pumps such as QepA and OqxAB, which actively expel fluoroquinolones from bacterial cells (14, 17).

Despite extensive studies on fluoroquinolone resistance in A. baumannii, there remains a critical gap in understanding the potential for restoring antibiotic susceptibility through efflux pump inhibition. Efflux pumps significantly contribute to resistance by expelling antibiotics before they reach their intracellular targets, thereby reducing their effectiveness (18, 19). Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) is a known efflux pump inhibitor that disrupts the proton motive force across bacterial membranes, potentially restoring antibiotic efficacy (20, 21). However, while CCCP has been explored in other bacterial species, its role in reversing fluoroquinolone resistance in A. baumannii remains underexplored.

This study aims to explore the prevalence and mechanisms of fluoroquinolone resistance in A. baumannii strains isolated from hospitals in Hamadan, as well as the effects of CCCP on the susceptibility of A. baumannii to fluoroquinolones.

In 2019, the World Health Organization (WHO) ranked antibiotic resistance among the top ten threats to public health globally, emphasizing its serious implications (31). One of the key pathogens contributing to this issue is A. baumannii, a gram-negative coccobacillus that is commonly associated with hospital-acquired infections. This bacterium’s ability to survive for long durations in healthcare environments, along with its resistance to multiple antibiotic classes, plays a significant role in the occurrence of outbreaks. Our study highlights this concerning situation, as we found high levels of antibiotic resistance among 102 A. baumannii isolates from Hamadan Regional Hospital in Iran, with 87 identified as MDR.

Fluoroquinolones has been considered as a viable treatment option when A. baumannii isolates are sensitive to it, offering a potentially preferable alternative to carbapenems in such cases (32). However, many A. baumannii strains resistant to ciprofloxacin also exhibit resistance to various other antibiotic classes. Consequently, alternative treatment strategies, such as carbapenem-sulbactam combinations, colistin, or tetracyclines, are often necessary. Assessing ciprofloxacin resistance is crucial in guiding the appropriate selection of therapeutic interventions for infections caused by A. baumannii (33, 34). A significant mechanism contributing to resistance against fluoroquinolones, including ciprofloxacin, and other antibiotics like tetracycline, involves the AdeABC efflux pump system. These efflux pumps actively remove various compounds, including antimicrobial agents, from bacterial cells, thereby facilitating resistance (35, 36). The overexpression of these chromosomal efflux systems, particularly in gram-negative bacteria, is a common driver of multidrug resistance and is associated with the RNA superfamily (37).

Our study revealed a notable presence of PMQR genes among A. baumannii isolates, with the qnrS gene detected in 77.45% of isolates, oqxA in 73.52%, qnrA in 72.54%, oqxB in 34.31%, and qepA in 11.76%. Interestingly, none of the isolates harbored the qnrB or qnrC genes. These findings are largely in line with those of Mohammed et al., who also reported high rates of PMQR genes in ciprofloxacin-resistant isolates (38). In their study, qnrA was found in 66.27% of isolates, qnrS in 70.93%, oqxA in 73.25%, and oqxB in 39.53%, with no detection of qepA or qnrB. Our study showed slightly higher prevalence rates for qnrA and qnrS, while the occurrence of oqxB was lower. These variations might reflect differences in resistance gene distribution across different regions or healthcare environments. In comparison, Ali et al. reported that 48.3% of A. baumannii isolates harbored one or more PMQR genes, a somewhat lower rate than that observed in our study (39). Yang’s study, on the other hand, found a much lower prevalence of PMQR genes among A. baumannii isolates. Only 7.7% of isolates carried qnrB, and 2.6% carried qnrS, highlighting a stark contrast to our findings. This significant difference could be due to regional variations in the spread of resistance genes or differences in the clinical settings from which the isolates were obtained (40). The lower detection rates in Yang’s study underscore the importance of considering geographic and environmental factors when evaluating antimicrobial resistance patterns.

Our PCR-RFLP analysis revealed that among the 102 A. baumannii isolates, 38 had mutations in the gyrA gene, 34 had mutations in the parC gene, and 22 exhibited mutations in both genes. These findings are consistent with those of Tawfick and El-Borhamy, who reported single mutations in either gyrA or parC in 11 isolates and dual mutations in the QRDRs of both genes in 38 isolates (41). However, our results differ from those of Zaki et al., who found that all ciprofloxacin-resistant A. baumannii strains carried mutations in either gyrA, parC, or both, with dual mutations being the most common (85.5%), and only 5% of strains showing mutations in a single gene (42).

It is well-established that mutations in both gyrA and parC are generally required for A. baumannii to develop high-level resistance to ciprofloxacin (43). In our study, however, we identified 12 ciprofloxacin-resistant and 17 levofloxacin-resistant isolates with single mutations in either gyrA or parC, yet these isolates still exhibited elevated MICs (≥ 128 μg/mL). This suggests that even single mutations in these genes may contribute significantly to resistance to ciprofloxacin and levofloxacin, which is consistent with the findings of Elshahat et al. (44).

Additionally, our study demonstrated that isolates harboring dual mutations in both gyrA and parC genes had notably higher MIC values. All such isolates had MICs exceeding 32 μg/mL for ciprofloxacin and 64 μg/mL for levofloxacin, with the majority of double-mutant isolates showing MICs above 128 μg/mL. These results are in line with studies by Ardebili et al. and Taha et al., who reported that strains with mutations in both gyrA and parC exhibited much higher resistance to ciprofloxacin compared to those with mutations in just one gene (28, 45).

The use of efflux pump inhibitors represents a significant advancement in combating antibiotic resistance (46). This study examined the effect of CCCP on enhancing the susceptibility of A. baumannii to ciprofloxacin and levofloxacin. Notably, following the addition of CCCP, 6.38% of the isolates that were previously resistant to ciprofloxacin became sensitive, while 8.82% of the levofloxacin-resistant isolates also showed increased susceptibility. Previous studies have shown that efflux pump inhibitors can significantly reduce multidrug resistance rates in A. baumannii and other Gram-negative bacteria. For instance, in the work of Carbonel et al., the addition of CCCP led to 6.38% of imipenem-resistant isolates becoming sensitive to cefepime, with a total of 17.02% of isolates demonstrating increased susceptibility (20). Similarly, Lin et al. reported comparable enhancements in ciprofloxacin susceptibility when CCCP was used (47). Our findings further revealed that 17.64% of isolates exhibited a 4-fold or greater reduction in the MIC for ciprofloxacin, while 12.74 % of isolates showed a similar reduction for levofloxacin. Approximately half of the strains demonstrated a 2-fold dilution reduction in MIC for both antibiotics. Notably, only one strain experienced a 2-fold increase in MIC for levofloxacin. Although our findings are encouraging, other research has reported even greater decreases in MIC values when using efflux pump inhibitors. Gholami et al. showed that the use of Phenylalanine-Arginine β-Naphthylamide led to a 4- to 64-fold decrease in MIC in 96.60% of the 60 A. baumannii isolates examined (29). Additionally, Ardebili et al. observed that the susceptibility of A. baumannii to ciprofloxacin improved significantly with CCCP treatment, resulting in MIC reductions of 2–64 dilutions for 86.1% of isolates (48). Furthermore, Rajamohan et al. reported that the addition of CCCP caused a 2- to 12-fold decrease in the MIC of various biocides compared to their initial MIC levels (49).

In our study, the expression levels of qepA and oqxA were evaluated under different conditions, including treatment with ciprofloxacin and levofloxacin, both alone and in combination with CCCP. Our results showed that when CCCP was combined with these antibiotics, it reduced the expression of qepA and oqxA. However, the most significant reduction in gene expression occurred when the antibiotics were used alone, indicating that the presence of CCCP partially counteracted this effect. Previous studies have demonstrated that CCCP, as an efflux pump inhibitor, influences gene expression in various bacterial species. Rafiei et al. reported that CCCP significantly decreased adeB efflux pump gene expression in A. baumannii when combined with tigecycline, suggesting its potential in modulating resistance across different antibiotics (50). Similarly, Baron et al. found that CCCP reduced mcr-1 expression in Gram-negative bacteria, a gene linked to colistin resistance (51). One possible explanation for our findings is that CCCP, by disrupting the proton motive force, alters bacterial stress responses, leading to regulatory shifts in efflux pump gene expression (52). While CCCP is expected to inhibit efflux activity, the partial reversal of antibiotic-induced gene suppression may indicate a compensatory bacterial mechanism aimed at maintaining efflux function despite the inhibitor’s effects. Further research is needed to clarify these regulatory interactions and their implications for resistance management.

The high prevalence of multidrug-resistant A. baumannii isolates in our regional hospital highlights the urgent need for effective antimicrobial strategies. Our findings underscore the significant role of PMQR genes and mutations in gyrA and parC in driving fluoroquinolone resistance. Although efflux pump inhibitors like CCCP show promise in enhancing antibiotic susceptibility, persistent resistance remains a major challenge. To translate these findings into clinical practice, further research is essential. While this study provides valuable insights, certain limitations must be acknowledged. Conducted in a single regional hospital, the findings may not be fully generalizable to other healthcare settings. Although the sample size was adequate for meaningful analysis, larger multicenter studies would improve the robustness of these conclusions. Additionally, our study primarily focused on genetic resistance determinants; functional assays evaluating efflux pump activity and other resistance mechanisms could further clarify the observed resistance patterns. Future research should prioritize clinical trials assessing the efficacy and safety of CCCP and other efflux pump inhibitors in combination with antibiotics. Moreover, given the clinical importance of antibiotics such as ampicillin-sulbactam and meropenem in the treatment of A. baumannii infections, future studies could explore the potential role of efflux pump inhibitors like CCCP as adjunctive agents to enhance the efficacy of these therapies. Investigating such combinations may open new avenues for overcoming resistance and improving patient outcomes in the face of limited treatment options. Investigating such combinations may open new avenues for overcoming resistance and improving patient outcomes in the face of limited treatment options. Furthermore, investigating novel efflux pump inhibitors and alternative combination therapies may offer promising treatment options for A. baumannii infections. Continued surveillance of resistance patterns and the development of targeted therapeutic strategies remain essential to combat this growing public health threat.