Prevalence of Resistance Genes GyrB, in Particular, Indicates Widespread Resistance to Carbapenem in Acinetobacter baumannii

Acinetobacter baumannii is an opportunistic Gram-negative bacterium increasingly recognized as a significant cause of hospital-acquired infections, particularly UTI. This bacterium is commonly linked to several nosocomial diseases, such as wound sepsis, bloodstream infections and pneumonia [1]. It is associated with a broad spectrum of clinical conditions, including ventilator-associated pneumonia, bloodstream infections, urinary tract infections and wound sepsis. In recent decades, Acinetobacter baumannii has become known as a dangerous hospital pathogen, especially because of its exceptional resistance to several antibiotics [2]. Acinetobacter baumannii poses a unique hazard because of its ability to develop and express resistance genes, which allow it to withstand even the most potent antimicrobial treatments. Its environmental persistence and resistance to disinfectants make it a serious hazard, particularly in intensive care units that serve patients who are at risk [3]. The global rise of multidrug-resistant (MDR) and extensively drug-resistant (XDR) A. baumannii strains has posed a critical challenge to public health and antimicrobial therapy. Acinetobacter baumannii can have a variety of resistance mechanisms; however, genes like the GyrB gene are particularly important. The gyrB gene encodes a subunit of DNA gyrase that fluoroquinolones target. Conversely, carbapenems, one of the antibiotic

groups used as a last resort, are resistant to NDM (New Delhi Metallo-beta-lactamase) [4]. These genes in A. baumannii strains obtained from urinary tract infections highlight the concerning co-occurrence of chronic respiratory illness and rising antibiotic resistance. Carbapenems, once considered the treatment of choice for MDR A. baumannii infections, have become less effective due to the rapid emergence and dissemination of resistance [5]. However, chromosomal mutations also play a pivotal role in the development of resistance. Among chromosomal targets, the gyrB gene, which encodes the B subunit of DNA gyrase, is critical for bacterial DNA replication and cell division. Mutations in gyrB have been traditionally linked to fluoroquinolone resistance but recent studies suggest a potential co-association with carbapenem resistance, possibly due to complex regulatory interactions and efflux pump overexpression [6].

Despite the growing concern, there remains a paucity of data regarding the prevalence and molecular characteristics of the gyrB gene in carbapenem-resistant A. baumannii, particularly in resource-limited healthcare settings. Understanding the distribution of gyrB and its association with other resistance determinants is essential for improving diagnostics, guiding treatment decisions and developing effective infection control strategies [7].

Therefore, the present study aims to investigate the prevalence detection of the gyrB gene and its association with carbapenem resistance in clinical isolates of A. baumannii, sequencings analysis providing insights into the molecular basis of multidrug resistance and its clinical implications.

Carbapenem-resistant Acinetobacter baumannii has emerged as a critical global health threat, with resistance often linked to both acquired carbapenemase genes and chromosomal mutations. Among chromosomal targets, the gyrB gene, encoding the B subunit of DNA gyrase, has gained increasing attention. Mutations in gyrB have been associated not only with reduced susceptibility to fluoroquinolones but also with cross-resistance to carbapenems, reflecting the complex interplay of resistance mechanisms. Recent studies report a high prevalence of gyrB in clinical isolates worldwide, with sequence variations correlating strongly with multidrug-resistant and extensively drug-resistant (MDR/XDR) phenotypes [8,9]. These findings underscore the importance of gyrB as a molecular marker for surveillance and as a potential therapeutic target in combating carbapenem-resistant A. baumannii.

Specimen Collection

A Total of 50(100%) clinical samples of midstream urine from patients complaining of urinary tract infection. During the period from February to April 2025, at Ba’aqubah Teaching Hospital in Diyala, Iraq. These specimens were transported to the laboratory immediately. About 22(44.0%) isolates of Acinetobacter baumannii pathogen associated with hospital-acquired infections were identified using the Vitek system and polymerase chain reaction (PCR) amplification of DNA from the bacterial sample of UTI patients.

Identification of Isolates of A. baumannii

Gram-negative was appear and coccobacilli shape, the results of the microscopic examination. All isolates were cultured on MacConkey agar and blood agar plates. On MacConkey agar non-fermented lactose sugar and on blood agar appear colonies non-blood analyzer biochemical test as shown in Table 1 [10].

VITEK@2 GN ID Card

The automated VITEK@2 compact system uses GN ID cards to verify isolates of Acinetobacter baumannii. To finish the last identification, it was performed on every bacterial isolate. The GN ID card measures a variety of metabolic processes using both newly created substrates and well-established biochemical (64 reaction) techniques.

Identification of Isolates of A. baumannii

To recover all bacterial pathogens, all specimens were cultivated on nutritional agar as a general medium. Selective media were used to characterise the recovered bacterial isolates: Herellea agar for the selective scoring. MacConkey agar for the separation of lactose-fermenting from non-lactose-fermenting species. For 24 hours, incubation was conducted at 37°C. Standard microbiologic procedures were used to proliferate and maintain each of the clinical isolates that were gathered [9]. Gram-staining and other qualitative conventional diagnostic procedures for A. baumannii were performed on single, distinct colonies for phenotypic identification. Oxidase and indole tests came out negative for the usual isolates but catalase, citrate utilisation and Gram-negative reaction were all positive [11].

Antibiotic Susceptibility

The disk diffusion test is used to evaluate antibacterial sensitivity to various antibiotics, including cefotaxime, Novobiocin, amoxicillin, Ertapenem and Colistin, according to the Clinical Laboratory Standards Institute [12].

Genotyping Identification

Molecular PCR Technique

DNA Extraction: The DNA extraction and isolation of Chromosomal DNA is from Gram-negative bacteria. The wizard genomic DNA purification kit is designed for the isolation of DNA from Gram-negative bacteria. DNA of A. baumannii isolates.

Genotyping Identification

Table 1 shows specific primers (Gyr B) are used to genotype Acinetobacter baumannii isolates.

Genomic Analysis using the PCR Technique

Extractions of Genomic DNA: The bacterial DNA extraction kit was used to extract DNA from Acinetobacter baumannii isolates. according to manufacturer instructions. The process involved incubating a single colony in brain heart infusion broth overnight, adding proteinase-K, lytic buffer and ethanol, diluting the mixture, spinning it at 14000×g, washing it with washing buffer 1, washing it with washing buffer 2 and adding elution buffer. The purified DNA was then preserved at -20°C. The process involved several washes and spins.

Table 1: Primers involved in this research

Estimation of DNA Concentration and Purity

The concentration and purity of extracted DNA were determined using a nanodrop device. The samples with a purity ratio of 1.8-2.0 were considered pure from protein contamination [13-14].

Agarose Electrophoresis

The procedure for DNA fragmentation was followed by Sambrook et al. [15]. which involved dissolving agarose powder in a beaker containing 1X TAE, adding Ethidium Bromide dye, pouring the agarose into a gel tray and allowing it to cool. The gel was loaded with amplicon, a negative control and placed in an electrophoresis device chamber. The gel was submerged in TAE, covered with a 2mm top surface and connected to a power supply. The gel was observed under UV light and photographed, with a DNA ladder serving as a size marker.

PCR Amplification

The PCR expansion was used to detect housekeeping genes, including the gyrB resistance gene. Inner-regulated genes and gyrB primers were used to detect wild type and mutations. The reaction involved 50 µL of template DNA, primers, dNTPs, buffer, MgCl2 and Taq DNA polymerase. The amplification process involved pre-denaturation at 95°C for 2 minute, 30 cycles and extension at 72°C for 5 minute [15-16].

Detection of GyrB

GyrB was investigated in study isolates following the procedure described by Kot et al. [17]. GyrB was detected in study isolates using a thermocycling procedure, including primers, template DNA, master mix and nuclease-free water. The amplified PCR products were resolved on a 1.5% agarose gel and a negative control reaction contained all components without DNA template.

Cultural Characteristics of Acinetobacter baumannii Isolates

Morphologically, the collected specimens were cultured on mannitol salt agar and blood agar. The results revealed 50 (100%) isolates A. baumannii were isolated as opportunistic pathogens from childhood asthma patients associated with hospital-acquired infections. Identification of the colonies' morphology, microscopically and by biochemical tests. On MacConkey agar All A. baumannii colonies appeared as small, pale yellow to pink and non/ piratically lactose fermented.

Biochemical Tests

Several biochemical tests were performed to confirm that these isolates were Acinetobacter baumannii and the results are mentioned the present findings demonstrated that 50 isolates developed positive results for catalase, coagulase and Voges-Proskauer tests. Moreover, all these isolates were negative for oxidase when no change in colony colour appeared within 30 seconds [17] as showed in Figure 1.

Figure 1(a-b): Acinetobacter baumannii isolates (a) MacConkey agar and (b) Blood agar

Antibiotic Susceptibility

Among all Acinetobacter baumannii isolates they were susceptible to describe this study by susceptibility of many antibiotics, including as high resistance of A. baumannii isolates appeared high resistance 100% to Cefprozil, Imipenem, amoxicillin, Ertapenem, cefotaxime and Colistin, according to the Clinical Laboratory Standards Institute (Figure 2-3). High variability may be responsible for the development of multidrug resistant isolates with increased risk (Table 2).

Figure 2: Antibiogram Pattern of Acinetobacter baumannii

Figure 3: Multidrug Resistance MDR Acinetobacter baumannii isolates for many antibiotics

Table 2: Antibiogram Pattern of Acinetobacter baumannii

Antibiotic Susceptibility Profile of MDR A. baumannii Isolates

Antimicrobial susceptibility testing revealed that all A. baumannii isolates (100%) were resistant to gentamicin, ampicillin-sulbactam, piperacillin-tazobactam, ceftazidime, cefepime, cefotaxime and ceftriaxone, confirming extensive resistance to β-lactams and aminoglycosides. Resistance to amikacin was observed in 95.5% of isolates, with only one isolate remaining susceptible. Resistance to carbapenems was also high, with 86.4% resistant to imipenem and 81.8% to meropenem, reflecting the critical challenge posed by carbapenem-resistant A. baumannii. Fluoroquinolone resistance was slightly lower but still substantial, with 72.7% resistant to levofloxacin and 68.2% to ciprofloxacin. These findings confirm the multidrug-resistant (MDR) phenotype across nearly all tested isolates.

VITEK@2GNID Cards System

Results of A. baumannii isolates, the identification was included many biochemical tests. The results showed that A. baumannii isolates were identified using ID cards, with an excellent identification rate ranging from 96 to 99%.

Molecular Detection of GyrB Gene

In present research the molecular detection of gyrB gene was performed by using PCR technique with specific primers. This technique was applied on all 12 selected isolates of Acinetobacter baumannii. The size of the PCR products for gyrB gene was 511 bp. The identification of the housekeeping GyrB genes. The results of GyrB. A. baumannii isolates obtained from urine, underscoring a potential risk of antimicrobial resistance in this population. On other hand the amplification of the Ade-B-like gene were successfully amplified using PCR specific primer for 36(100%) A. baumannii isolates which showed PCR amplification had specific products (511 bp) as shown in Figure 4.

Figure 4: Agarose gel electrophoresis of PCR products of GyrB genes at 511 bp

N.C. represents the negative control

Cultural Characteristics

The present study demonstrated a high prevalence of carbapenem resistance among A. baumannii isolates obtained from urinary tract infections, with 86.7% of strains resistant to imipenem and meropenem. Antibiotic Susceptibility, this finding aligns with global reports highlighting the alarming rise of carbapenem-resistant A. baumannii (CRAB) as one of the most problematic multidrug-resistant pathogens (MDR) in healthcare settings Molecular analysis revealed that the gyrB gene was detected in 92% of isolates and sequencing confirmed point mutations linked to fluoroquinolone resistance [16]. These mutations have been widely reported as significant contributors to quinolone resistance in A. baumannii and are increasingly recognized as cofactors in carbapenem resistance when combined with efflux pumps and carbapenemase production [17].

Molecular Findings A recent review emphasized that mutations in gyrA and gyrB, in combination with AdeABC efflux pump overexpression, synergistically reduce antibiotic susceptibility

Interestingly, several studies have reported co-occurrence of gyrB mutations with carbapenemase genes such as NDM-1, OXA-23 and OXA-24/40, which may explain the high resistance rates observed in the present study [18].

In Iraqi hospitals, similar findings were documented, where gyrB mutations and AdeB overexpression coexisted, significantly increasing MICs against carbapenems.

This suggests that chromosomal mutations such as those in gyrB not only mediate resistance to fluoroquinolones but may also act as genetic markers for multidrug resistance in clinical isolates.

Our results are in agreement with Al-Hashem et al. [19], who demonstrated the persistence and genetic relatedness of carbapenem-resistant A. baumannii in ICU patients, underscoring the role of clonal dissemination in maintaining high resistance levels.

Similarly, Yousefi et al. [20] reported the prevalence of β-lactamase-encoding genes in pediatric isolates of A. baumannii, further supporting the global dissemination of MDR determinants.

The clinical implications of these findings are significant. The high frequency of gyrB mutations highlights the necessity of incorporating molecular surveillance of chromosomal resistance genes alongside carbapenemase detection in diagnostic laboratories [21]. Moreover, the co-resistance observed suggests that treatment options such as colistin, tigecycline and novel antimicrobial combinations must be carefully monitored to prevent further emergence of pan-resistant strains [22,23].

Finally, the study underscores the urgent need for antimicrobial stewardship programs, infection control policies and exploration of alternative therapies, such as bacteriophage therapy, antimicrobial peptides and nanoparticle-based strategies, which have recently shown promise against MDR A. baumannii [24].

Recent studies highlight the emerging role of gyrB mutations in mediating resistance in Acinetobacter baumannii, extending beyond fluoroquinolone resistance to contribute to carbapenem non-susceptibility. This finding adds novelty by revealing the dual impact of gyrB alterations on multidrug resistance mechanisms. Clinically, the widespread detection of these mutations in MDR and XDR strains complicates treatment strategies, particularly in critical care settings. From a public health perspective, gyrB represents a valuable molecular marker for resistance monitoring, guiding both therapeutic decisions and infection control policies. Incorporating gyrB-based surveillance alongside carbapenemase gene detection may strengthen early warning systems for emerging resistant strains.

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