Original Article

Genotypic Investigation of Antibiotic Resistance: Presence of BlaOXA-48 Gene in Clinical Isolates of Pseudomonas aeruginosa

Please cite this article as follows: Pourmohammadi N, Shapouri R, Nahaei M, Mohammadi Roknabadi M. Genotypic investigation of antibiotic resistance: presence of BlaOXA-48 gene in clinical isolates of Pseudomonas aeruginosa. Avicenna J Clin Microbiol Infect. 2024; 11(2):62-67. doi:10.34172/ajcmi.3508

Introduction

Pseudomonas aeruginosa is a pathogenic and opportunistic bacterium, which is a major factor in the death of patients with weakened immune systems. Its intrinsic resistance to antimicrobial substances worsens the treatment of infections caused by this bacterium (1,2). Lipopolysaccharides, pili, and polar flagellum in this opportunistic pathogen facilitate its adherence to the host cell membrane and play an important role in its pathogenicity (3). Beta-lactams are effective drugs for treating P. aeruginosa, but over time, some bacteria show resistance to this antibiotic by producing beta-lactamase enzymes (4).

Hospital infections are one of the major medical problems in both developed and developing countries, leading to the spread of infectious diseases in society (5). In recent years, there has been an increasing focus on hospital infections because they cause thousands of deaths worldwide (6). P. aeruginosa is one of the most common causes of hospital-acquired infections, which can induce septicemia, pneumonia, meningitis, and other fatal diseases (7). P. aeruginosa has an innate resistance to a wide range of antimicrobial and disinfectant substances such as ammonium compounds, hexachlorophene, soaps, and iodine solutions (8). Furthermore, most antibiotic-resistant strains can produce a wide spectrum of beta-lactamase enzymes (8).

The emergence of resistance in this bacterium is caused by several mechanisms, including the activation of efflux pump, the production of beta-lactamase enzymes, and changes in the outer membrane containing group B carbapenemases (metallo-beta-lactamases) such as Verona integron metallo-beta-lactamase (VIM) and imipenemase (IMP) enzymes (9,10). Metallo-beta-lactamases, which need a cofactor such as zinc for their activity, can hydrolyze not only carbapenems but also all beta-lactam antibiotics (11). The resistance of P. aeruginosa strains to second- and third-generation (broad-spectrum) cephalosporins and monobactams may be due to the production of extended-spectrum beta-lactamase (ESBL) enzymes (12,13). These enzymes are encoded by different genes located on the chromosome or plasmid and open the beta-lactam ring, thereby inactivating the antibiotic (14).

Given the importance of P. aeruginosa in hospital infections and the lack of knowledge about the frequency of ESBLs in clinical samples, this study was conducted to determine the phenotypic and genotypic prevalence of ESBL-producing P. aeruginosa strains in different clinical samples from Asadabadi hospital, Tabriz, Iran.

Materials and Methods

Collection of Isolates

This descriptive-analytic study involved collecting 100 clinical isolates of P. aeruginosa from various clinical samples, including urine, burn wound, tracheal tube, and blood, from patients referred to clinical laboratory in Asadabadi hospital, Tabriz, Iran, during 2020-2021. Biochemical analysis such as hemolysis, urea production, oxidase activity, H2S production, indole, Voges–Proskauer, catalase activity, citrate utilization, and gram staining were used for the primary identification of the isolates. The identified isolates were frozen in Tryptic Soy Broth (TSB) containing 15% glycerol at -70 °C for subsequent analysis.

Antibiotic Susceptibility Analysis

The antibiotic susceptibility of the isolates was evaluated using the disk diffusion method as described by the Clinical and Laboratory Standards Institute (CLSI) (15). This method involves placing various antibiotic-impregnated disks on agar plates previously inoculated with a bacterial suspension. The antibiotics diffuse radially outward through the agar, creating a concentration gradient. After 24 hours of incubation at 35 ± 1 °C, the diameters of the inhibition zones around each disk were measured visually. The antibiotic discs included cefepime (30 µg), imipenem (30 µg), tobramycin (5 µg), colistin (10 µg), ceftazidime (30 µg), amikacin (30 µg), piperacillin (100 µg), ciprofloxacin (5 µg), and gentamicin (10 µg). Moreover, the antibiotic susceptibility of P. aeruginosa ATCC 27853 was evaluated as a control.

Multidrug-Resistant Classification

Isolates demonstrating simultaneous resistance to at least three antibiotics were categorized as multidrug-resistant (MDR) (16).

Phenotypic Detection of Extended-Spectrum Beta-Lactamase-Producing Isolates

Phenotypic detection of the ESBL-producing isolates was conducted through the combination disc method. The antibiotic discs included cefotaxime (30 µg), ceftazidime (30 µg), cefotaxime-clavulanic acid (30 μg-10 μg), and ceftazidime-clavulanic acid (30 µg-10 µg). An isolate was considered ESBL-producing if the diameter of the growth inhibition produced by combined discs was greater than the diameter of the growth inhibition halo produced by the same discs alone(16).

Genotypic Detection of Extended-Spectrum Beta-Lactamase-Producing Isolates

The genomic DNA was extracted from the ESBL-producing P. aeruginosa isolates using a specific kit (Invitek Stratec Business, Canada) according to the manufacturer’s instructions. Molecular detection of the blaOXA-48 gene in DNA samples of the isolates was conducted by polymerase chain reaction (PCR) method in a 25 µL total volume (1 µL DNA sample, 1 µL each primer, 12.5 µL master mix, and 9.5 µL deionized water). The PCR experiment commenced with an initial denaturation cycle, which involved subjecting the sample to a temperature of 95 °C for 5 minutes, followed by 45 denaturation cycles at 94 °C, each lasting 30 seconds. Subsequently, the annealing procedure was carried out at 50 °C for 30 seconds, followed by an extension at 72 °C for 1 minute. Ultimately, a final extension cycle was executed at 72 °C for 5 minutes (16). The primer sequences employed in this study were as follows: forward 5΄-GCTTGATCGCCCTCGATT-3΄ and reverse 5΄-GATTTGCTCCGTGGCCGAAA-3΄. The amplified PCR product, with a length of 830 base pairs, was estimated by electrophoresis on a 2% agarose gel. P. aeruginosa ATCC 27853 was used as control.

Statistical Analysis

The raw data were analyzed by SPSS statistical software (version 16) and Excel software (version 2010). The association of antibiotic resistance with the presence of the blaOXA-48 gene was analyzed using Fisher’s exact test and the chi-square (χ²) test. The statistical significance level was considered at P value < 0.05.

Results

Bacterial Isolates

This study thoroughly investigated 622 samples from various clinical sources, including urine, burn wounds, tracheal tubes, and blood. Of these, 100 clinical isolates of P. aeruginosa were successfully isolated and identified using biochemical analytical techniques. Out of 100 P. aeruginosa isolates, 18 (18%) were isolated from urine samples, 33 (33%) from burn wound samples, 26 (26%) from tracheal tube samples, and 23 (23%) from blood samples.

Antibiotic Resistance Patterns

The clinical isolates indicated a high rate of resistance to ceftazidime (89%), ciprofloxacin (83%), and tobramycin (81%) antibiotics. However, a high rate of sensitivity was observed to colistin (81%), amikacin (65%), and gentamicin (64%) antibiotics (Table 1).

Phenotypically Detected Extended-Spectrum Beta-Lactamase-Producing Isolates

Phenotypic analysis indicated that 68 (68%) out of the 100 clinical isolates of P. aeruginosa were ESBL-producing. Of these 68 ESBL-producing isolates, 14 were from urine samples, 23 were from burn wound samples, and 14 were from tracheal tube samples (Table 2).

Genotypically Detected Extended-Spectrum Beta-Lactamase-Producing Isolates

Genotypic analysis indicated that 41 (60.30%) out of the 100 isolates carried the blaOXA-48 gene and were considered ESBL-producing isolates (Table 3). The statistical analysis revealed a significant association between the presence of the blaOXA-48 gene and P. aeruginosa isolates’ resistance to cefepime, imipenem, and gentamicin (Table 4).

Discussion

The management of P. aeruginosa infections is difficult due to its inherent resistance to multiple antimicrobial agents through several mechanisms. These bacteria become resistant to beta-lactam antibiotics by producing beta-lactamase enzymes (17). Metallo-beta-lactamase enzymes, a type of beta-lactamases, cause resistance to most beta-lactam antibiotics (18). The rapid transmission and spread of P. aeruginosa strains capable of producing these enzymes have led to an increase in the number of hospital infections worldwide (19). P. aeruginosa is the third cause of hospital infections, following Staphylococcus aureus and Escherichia coli (20).

In the current study, 100 P. aeruginosa strains were isolated from urine, burn wounds, tracheal tubes, and blood samples, with 68 (68%) isolates identified as ESBL-producers. These strains indicated high resistance to antibiotics such as tobramycin (81%), ciprofloxacin (83%), and ceftazidime (89%), whereas the lowest resistance was related to gentamicin (18%). Moreover, the blaOXA-48 gene was found in 41 (60.30%) of the ESBL-producing isolates.

Antibiotic resistance is an important global problem, thus numerous studies have been conducted on ESBL-producing P. aeruginosa isolates (21). In a study conducted by Fazeli et al in Isfahan, 94% of P. aeruginosa clinical isolates were resistant to imipenem, piperacillin, and ciprofloxacin antibiotics, with all strains showing resistance to ceftazidime and ticarcillin antibiotics (22). In a study on P. aeruginosa isolates from patients in the burn center of Tehran, Ranjbar et al reported that 97% of strains are resistant to imipenem, and 90% of strains are resistant to amikacin (23). In another study, Salehi et al reported 86% resistance of P. aeruginosa to nalidixic acid and 79% resistance to ceftazidime (24). Mihani et al found a 71% resistance rate to ceftazidime (25). Previous studies reported a significant relationship between the use of antibacterial drugs (e.g., amikacin, ciprofloxacin, ceftazidime, and imipenem) and the spread of resistant P. aeruginosa strains (26,27). Sadredinamin et al identified 95 imipenem- and meropenem-resistant strains from 100 P. aeruginosa isolates, with 81 isolates being ESBL-producing strains (28). Likewise, Fallah et al found that out of 100 P. aeruginosa strains isolated from clinical samples, 83 were resistant to imipenem, and 48 were ESBL-producers (29). These studies collectively indicate that antibiotic resistance in P. aeruginosa is pervasive and multifaceted, necessitating comprehensive strategies to address this issue. Potential solutions include the development of new antibiotics, the use of combination therapies to counteract resistance mechanisms, and the implementation of stringent antibiotic stewardship programs to reduce the misuse and overuse of existing antibiotics. Additionally, increased surveillance and molecular studies are essential for understanding the mechanisms of resistance and tracking the emergence and spread of resistant strains. By addressing these challenges through a multi-pronged approach, we can better manage and mitigate the impact of antibiotic-resistant P. aeruginosa on public health.

Due to the genetic nature of P. aeruginosa, it accepts all types of genes through plasmids and transposons, which may cause the rapid resistance of this bacterium to various antibiotics (30). Our results demonstrated that the frequency of the blaOXA-48 genein ESBL-producing P. aeruginosa isolates is 60.30%, with a significant association between the presence of this gene and resistance to cefepime, imipenem, and gentamicin. This gene has been detected in different geographical areas worldwide, including Turkey, India, Afghanistan, China, the Middle East, and African countries (31). Begum and Shamsuzzaman in Bangladesh detected the OXA-48 gene in 20% of P. aeruginosa isolates (32). Mohamed et al in Sudan reported a 22.4% frequency of OXA-48 in clinical isolates of P. aeruginosa (33). In another study in Sudan, Ali and Nagla found a 60% frequency of OXA-48 in clinical isolates of P. aeruginosa, with a significant association between the presence of blaOXA-48 and resistance to carbapenems (34). In a study by Al-Alaq et al in Iraq, the frequency of the OXA-48 gene in 200 P. aeruginosa isolates obtained from the microbiology laboratory of the burn ward was 10% (35). Bahrami et al reported a 12.5% frequency of blaOXA-48 in Isfahan, which is lower than that in our study (36). In Mashhad, Hashemi et al found that 46.34% of 82 P. aeruginosa isolates were resistant to imipenem, with the highest resistance rate in carbenicillin (69.5%). All imipenem-resistant P. aeruginosa isolates were confirmed by multiplex PCR using primers that targeted the rpoD gene. Additionally, in multiplex PCR, among these imipenem-resistant isolates, 23.6% carried the blaOXA-48 gene (37). Interestingly, a common finding in previous studies is the increasing antibiotic resistance of P. aeruginosa isolates worldwide.

Limitations of the Study

To confirm isolates definitively, molecular tests are recommended. Although a series of biochemical assays, including Gram staining, urea synthesis, H2S generation, Voges–Proskauer, hemolysis, catalase, oxidase, and indole tests, were conducted, these tests are not specifically designed for the detection of P. aeruginosa. Therefore, alternative diagnostic tests should be employed to ensure accurate identification of P. aeruginosa. Additionally, although the study’s focus on the blaOXA-48 beta-lactamase gene and the antimicrobial resistance pattern provides valuable insights, the limited scope of the genetic analysis may overlook other relevant resistance mechanisms and genes present in the isolates. Future studies should consider comprehensive genomic analyses to capture a broader spectrum of resistance determinants.

Conclusion

This study revealed a high prevalence of ESBL-producing P. aeruginosa isolates in clinical samples. In addition, we assume that resistance of P. aeruginosa isolates to cefepime, imipenem, and gentamicin may be associated with the presence of the blaOXA-48 gene. However, further research is needed to assess the prevalence of ESBL-producing P. aeruginosa in other regions of Iran and to explore the frequency of other beta-lactamase genes in clinical samples to combat infections caused by MDR of P. aeruginosa.

Acknowledgments

This study was extracted from the Ph.D. project of Naser Pourmohammadi, advised by Dr. Reza Shapouri, Dr. Mohammadreza Nahaei, and Dr. Mojtaba Mohammadi.

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