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Edited by

Aiman ZahraVirtual University of Pakistan

Reviewed by

Susan Fawzi Khadhem Al-SudaniAl-Bayan University, Iraq

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  • Short Communication
  • Date Received: 04-09-2023
  • Date Revised: 14-10-2025
  • Date Accepted: 23-06-2026
  • Date Published: 26-08-2026

Molecular detection of efflux pump genes in clinical isolates of MDR Pseudomonas aeruginosa

Muslim M. Kadhim1, Aseel R. Mardan1
  1. Department of Biology, College of Education, University of Al-Qadisiyah, Iraq

Abstract

Background: Pseudomonas aeruginosa is an opportunistic pathogen and a leading cause of various infections, including community-acquired and healthcare-associated infections. This organism is highly resistant to different classes of antimicrobials through multiple mechanisms, presenting a major challenge for infection treatment in hospital settings. Therefore, this study aimed to detect RND-type efflux pump genes, including mexAB, mexXY, mexC, and oprM, in multi-drug resistant (MDR) P. aeruginosa clinical isolates from Diyala Province using PCR technology.

Methods: A total of 50 clinical isolates were recovered from patients with different infections between November 2022 and March 2023. Twenty MDR isolates were selected based on antimicrobial susceptibility testing and subsequently confirmed using the VITEK 2 Compact system. Demographically, the isolates were obtained from 23 males (46%) and 27 females (54%). Twenty MDR isolates were selected based on antimicrobial susceptibility testing against 10 distinct antimicrobials using the Kirby-Bauer disk diffusion method.

Results: Molecular screening revealed that 10/20 (50%) of the isolates harbored the mexB gene, while the mexA gene was not detected in any isolate (0/20, 0%). Notably, all isolates carried both the mexC and mexY genes (20/20, 100%). Furthermore, 2/20 (10%) of the isolates harbored the mexX gene, whereas only 1/20 (5%) was positive for the oprM gene.

Conclusion: The current study showed in conclusion a rise in the spread of Mex efflux pump genes between the MDR isolates of P. aeruginosa bacteria that we obtained from different clinical samples in Diyala province, and therefore the isolated positive efflux pumps were detected in this study and this indicates the extent of enhanced prevention and severe control of this infection.

Keywords

P. aeruginosa, Antimicrobial resistance, MDR, Efflux pump genes

Introduction

Pseudomonas aeruginosa (P. aeruginosa) is a highly pathogenic bacterium whose cell surface structures, secreted compounds, and biofilm formation all contribute to its virulence. In immunocompromised patients, it causes complex, difficult-to-treat infections owing to this virulence combined with antibiotic resistance [1]. As a quintessential opportunistic gram-negative pathogen, P. aeruginosa has been responsible for life-threatening infections in patients with weakened immune systems [2]. Resistance to antimicrobial agents in P. aeruginosa arises through three fundamental mechanisms: intrinsic, acquired, and adaptive resistance. Its inherent resistance stems from efflux pump expression, restricted outer membrane permeability, and the production of antibiotic-inactivating enzymes, while acquired resistance can develop through horizontal transfer of variable resistance genes, including β-lactamases, metallo-β-lactamases, and aminoglycoside resistance genes [3,4]. This study therefore screened for efflux pump genes (mexA, mexB, mexC, mexX, and mexY) and the outer membrane protein gene oprM.

P. aeruginosa is a Gram-negative bacillus widely distributed across environments, particularly in soil and water subject to heavy human activity, such as wastewater-, hydrocarbon-, and pesticide-contaminated sites [5]. It ranks among the leading pathogens responsible for nosocomial infections—affecting the respiratory tract, urinary tract, and skin wounds—and frequently causes recurrent, prolonged chronic infections in patients with cystic fibrosis [6,7]. As the body’s largest organ, the skin serves as a physical and immunological barrier against pathogenic microorganisms; cutaneous lesions therefore represent an entry point for microbial contamination that can progress to chronic wounds and other invasive infections. Given their substantial social, psychological, and economic burden, chronic wounds are regarded as a serious public health concern [8]. Infections caused by P. aeruginosa are particularly challenging because of both its intrinsic and acquired resistance to numerous otherwise effective antibiotic classes, with intrinsic multidrug resistance driven by limited outer membrane permeability, inducible β-lactamase production, and multidrug efflux systems [9]. This study aimed to detect the efflux pump genes mexA, mexB, mexC, mexX, and mexY, along with the outer membrane protein gene oprM, across a range of clinical isolates.

Methods

Confirmation of bacterial isolates                                                                               

A total of 50 P. aeruginosa isolates were obtained from various clinical samples collected from male and female patients of different ages admitted to Baquba General Hospital, the Burns Center, and private medical laboratories in Diyala Province. The 50 P. aeruginosa isolates were identified using macroscopic, microscopic, culture, and biochemical tests and confirmed by the VITEK 2 Compact system. Antimicrobial susceptibility testing was subsequently performed, from which 20 isolates demonstrated an MDR phenotype. The susceptibility of the P. aeruginosa isolates against 10 distinct antimicrobials was evaluated using the Kirby-Bauer disk diffusion method, confirming their multi-drug resistant (MDR) phenotype. Biofilm production was evaluated using the microtiter plate method, as previously described [10].

Bacterial DNA extraction and PCR 

Genomic DNA was extracted from the 20 MDR isolates using a commercial genomic DNA extraction kit according to the manufacturer’s instructions (Favorgen Biotech Corp., Korea). The extracted DNA was stored at -20 °C in a deep freezer until further amplification. PCR assays were performed to screen for all the target genes listed in Table 1. The amplified PCR products were separated via gel electrophoresis on a 1% agarose gel (iNtRoN Biotech Inc., Korea) stained with 0.5 µg/mL ethidium bromide. Band migration and amplification profiles were subsequently visualized and photographed using a gel documentation system (Cleaver, United Kingdom).

PCR amplification was performed under the following conditions: initial denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 54 °C for 30 s, and extension at 72 °C for 1 min, with a final extension at 72 °C for 7 min for all genes. Amplified PCR products were detected by agarose gel electrophoresis.

Results

Molecular Detection of Efflux Pump Genes (mexA, mexB, mexC, mexX, mexY and oprM)

All 20 MDR isolates (100%) demonstrated biofilm production capabilities using the microtiter plate method. The molecular analysis of efflux pump genes summarized in Table 2 revealed that 0/20 (0%) and 10/20 (50%) of the P. aeruginosa isolates were positive for the mexA and mexB genes, respectively. Additionally, PCR data revealed that 20/20 (100%) of the isolates harbored the mexC and mexY genes. Furthermore, 2/20 (10%) of the isolates harbored the mexX gene. However, PCR data for outer membrane genes showed that 1/20 (5%) of the isolates were positive for the oprM gene.

In this study, all isolates were tested for the presence of the mexA and mexB genes, and the mexA gene was not amplified in any isolate as shown in Figure 1. The mexA gene was not amplified in any of the tested isolates.

While the mexB gene was detected 10/20 (50%) as in figure (2). The molecular results of efflux pump genes in figure (3) revealed that 20/ 20 (100%) of P. aeruginosa isolates were positioned for mexC. Uniplex results by PCR technique, showed 20/20 (100%) of isolates were positive for mexY in (Figure 4). PCR data revealed that 2/20 (10%) of the isolates contained the mexX gene (Figure 5). The results of the present study demonstrated that only 1/20 (5%) of the MDR P. aeruginosa isolates harbored the oprM gene (Figure 6).

Discussion

The prevalence of efflux pump genes in the present study was generally lower than that reported in previous literature.

For mexA, Abbas et al., [12] reported a 100% detection rate among Pseudomonas aeruginosa isolates in Egypt, while Murugan et al., [13] detected it in 51% (or 54.2%) of isolates which are both markedly higher than the present study, in which the gene was not detected. Similarly, Al-Zowaid et al., [16] and Kishk et al., [17] reported higher mexA detection frequencies of 83.5% and 88.2%, respectively.

Regarding mexB, the current findings contrast with a Baghdad-based study where 100% of isolates carried the gene [15], as well as reports by Al-Zowaid et al., (63.29%) [16], Kishk et al., (70.5%) [17], and Murugan et al., (46.5%) [13]. Kishk et al., [17] also noted that 58.8% (80 strains) of isolates co-harbored both mexA and mexB. In contrast, our mexB findings are consistent with a study conducted in Iran, which observed the gene in 53.3% of isolates [14]. For the outer membrane gene oprM, the detection rate in this study was considerably lower than the 40.5% reported by Murugan et al., [13] and the 37.5% (12/32) reported by Al-Zowaid et al., [16].

Discrepancies were also evident across other efflux pump systems. The mexC gene was detected in 78.12% of isolates in a study conducted in Najaf City, Iraq [18], and a similarly high prevalence (89.47%) was reported by Al Saadi [19] in Diyala, both considerably higher than the rate observed in the current study. Conversely, Al-Jubori [20] identified mexY in 28.5% (8/28) of isolates in Iraq, representing a lower prevalence than that found in the present study.

The variation in efflux pump gene distribution across these studies likely reflects regional differences in multidrug-resistance patterns and the selective pressure driven by local antibiotic usage.

Conclusion

Statement & Declarations

Conflict of Interest

There is no conflict of interest to be declared by authors.

Author Contributions

Muslim M. Kadhim and Aseel R. Mardan contributed equally to conceptualization, execution and reporting of this study.

Acknowledgment

AI language tools were used in the introduction section to [only] improve readability of the text. 

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Article Sections

Edited by

Aiman ZahraVirtual University of Pakistan

Reviewed by

Susan Fawzi Khadhem Al-SudaniAl-Bayan University, Iraq

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Tables

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Edited by

Aiman ZahraVirtual University of Pakistan

Reviewed by

Susan Fawzi Khadhem Al-SudaniAl-Bayan University, Iraq

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Tables

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