Genotypic Investigation of Antibiotic Resistant blaOXA-4 Gene in Clinical Isolates of Pseudomonas aeruginosa

Background: Pseudomonas aeruginosa , an opportunistic Gram-negative bacterium, is responsible for 10-15% of hospital infections worldwide. The acquisition of resistance genes is one of the important mechanisms that causes the spread of resistance in this bacterium. This study aimed to conduct a phenotypic and genotypic investigation of the blaOXA-4 resistance gene in P. aeruginosa isolated from clinical samples. Methods: In this study, 110 P. aeruginosa strains were isolated from various clinical samples. The disk diffusion method was applied to reveal the resistance pattern in the isolates. Moreover, the combined disk method was used for the phenotypic analysis of extended-spectrum beta-lactamases (ESBL). Finally, the presence of the blaOXA-4 beta-lactamase gene was analyzed genotypically by polymerase chain reactions (PCR) method. Results: The highest sensitivity and resistance of the isolates were related to amikacin (65.45%) and ceftazidime (86.36%), respectively. The phenotypic analysis indicated that 72 isolates (65.45%) of P. aeruginosa are ESBL-producing. Furthermore, the presence of blaOXA-4 was approved genotypically in 33 P. aeruginosa isolates (45.83%). Conclusion: This study revealed a high prevalence of antibiotic-resistant isolates of P. aeruginosa in the East Azerbaijan population that may be associated with the presence of the blaOXA-4 gene. However, further studies are necessary to identify other resistant genes in ESBL-producing isolates and other geographical areas with larger sample size.


Introduction
Pseudomonas aeruginosa is the most pathogenic member of the Pseudomonadaceae family that includes gramnegative, non-fermenting, obligatory aerobic, oxidasepositive bacilli with mobility and growth ability in various environments (1,2).This bacterium is responsible for serious infections (e.g., otitis, keratitis, endocarditis, septicemia, and pneumonia) in the world that cause high rates of mortality in patients with neoplasmic disease, cystic fibrosis, and severe burns (3,4).
The spread of antibiotic-resistant strains of P. aeruginosa is the major cause of failure in infection control and the main reason for lethality in patients with defects in the immune system (5).This bacterium has an inherent resistance against various antiseptic and antimicrobial compounds that may be due to outer membrane impermeability to antibiotic penetration by active transmission (6,7).Furthermore, P. aeruginosa can acquire drug resistance through the increased production of secretory pumps as well as the production of carbapenamase and beta-lactamase enzymes (6,7).
Beta-lactam antibiotics family with beta-lactam rings are the most common antibacterial compounds worldwide that include carbapenems, monobactams, cephalosporins, and penicillin (8).Some bacteria produce beta-lactamase enzymes that cause the destruction or inactivation of betalactam antibiotics via hydrolysis central core of the betalactam ring and antibiotic resistance as a result (9,10).The production of new and widely used antibiotics such as broad-spectrum cephalosporins has led to the emergence of extended-spectrum beta-lactamases (ESBL) as a new class of beta-lactamases enzymes (11).Evidence suggested that Gram-negative bacteria, especially P. aeruginosa, encodes metallo-β-lactamase enzyme by several transferable genes (12,13).Therefore, the continuous increase in the prevalence of antibiotic-resistant strains has become an important concern (14,15).In the Ambler classification scheme, β-lactamases of classes A, C, and D are serine β-lactamases.In contrast, the class B enzymes are metallo-β-lactamases.Except OXA-type enzymes (which are class D enzymes), the ESBLs are of molecular class A. Moreover, class D enzymes such as OXA-type carbapenemases that can be encoded by chromosome or plasmid are proven to have a vital role in resistance to carbapenem (16,17).
Given the significance of increased antibiotic resistance in opportunistic microorganisms such as P. aeruginosa as well as the importance of resistance mechanisms knowledge to deal with infection by these bacteria, this study aimed to conduct a phenotypic and genotypic investigation of antibiotic resistance blaOXA-4 gene in clinical isolates of P. aeruginosa in East Azerbaijan population.

Collection of Isolates
We collected 110 P. aeruginosa isolates from different clinical sources such as burn wounds, tracheal tubes, urine, and blood samples of patients referred to Asadabadi hospital, Tabriz, Iran, during 2020-2021.Various standardized methods and biochemical analyses such as Gram staining, H 2 S production, urease production, Voges-Proskauer, hemolysis, catalase, oxidase, and indole were applied to identify the isolates.The approved isolates were then preserved by Tryptic Soy Broth (Merck, Germany) and 15% glycerol at -70 °C.

Genotypic Detection of Extended-spectrum Beta-lactamase -producing Isolates
The extraction of genomic DNA from the isolates was conducted by a specific kit (Invitek Stratec Business, Canada) according to instructions of the manufacturer.The polymerase chain reactions (PCR) method was applied for the genotypic detection of blaOXA-4 as a β-lactamases coding gene.Amplification was conducted in 25 µL total volume (12.5 µL master mixture, 1 µL each primer, 1 µL extracted DNA, and 9.5 µL sterile distilled water) followed by 1 cycle initial denaturation (95 °C for 5 minutes), 45 cycles denaturation (94 °C for 30 seconds), annealing (50 °C for 30 seconds), extension (72 °C for 1 minute), and 1 cycle final extension (72 °C for 5 minutes).The used primer sequence was forward 5΄-ATGAAAAACACAATACATATC-3΄ and reverse 5΄-TTATAAATTTAGTGTGTTTAG-3΄ with 830 bp product size.The amplified products were separated by electrophoresis on 2% agarose gel and photographed by gel document (Syngene, India), and P. aeruginosa ATCC 27853 was considered the control strain (9).

Statistical Analysis
The obtained raw data were analyzed statistically by SPSS statistical software (version 16).The association between antibiotic resistance and the presence of the blaOXA-4 gene was evaluated by Fisher and chi-square (χ2) tests.Moreover, the significance level was considered P value < 0.05.

Phenotypically Detected Extended-Spectrum β -Lactamase-Producing Isolates
The results of phenotypic detection of ESBL-producing P. aeruginosa demonstrated that out of 110 isolates, 72 cases (65.45%) are ESBL-positive.Moreover, out of 72 ESBLpositive isolates, 24 cases were from burn wound samples, 14 cases from tracheal aspirate samples, 17 cases from urine samples, and 17 cases from blood samples (Table 2).

Genotypically Detected Extended-Spectrum β-Lactamase-Producing Isolates
The results of genotypic detection of ESBL-producing P. aeruginosa demonstrated that out of 72 isolates, 23 cases (31.95%) carried the blaOXA-4 gene (Table 3).Additionally, the statistical analysis indicated that the presence of the blaOXA-4 gene is associated with resistance to cefepime antibiotic (Table 4).

Discussion
The high prevalence of P. aeruginosa with intrinsic resistance has led to the failure in the control and treatment of hospital infections by current antibacterial compounds.Therefore, the identification of antibiotic-resistant strains and various resistance factors provides a clear view for this problem (5).Resistant strains of P. aeruginosa commonly produce ESBL for the destruction the beta-lactam chain (18).In this regard, the wide spread of ESBL-producing bacterial strains, especially P. aeruginosa, has increased hospital infections worldwide (19).
In the present study, we evaluated the phenotypic and genotypic resistance of 110 P. aeruginosa strains isolated from clinical samples such as burn wounds, tracheal aspirate, urine, and blood.The results revealed that the highest antibiotic resistance of P. aeruginosa is related to ceftazidime (86.36%), ciprofloxacin (80.00%), and tobramycin (76.36%), respectively, whereas the highest sensitivity of the isolates was related to amikacin (65.45%).
So far, numerous similar studies have been reported in different geographic areas in Iran.Fazeli et al in Isfahan reported that all clinical isolates (100%) of P. aeruginosa are resistant to ticarcillin and ceftazidime (20).In another study, Ranjbar et al in Tehran demonstrated that all strains (100%) of P. aeruginosa isolated from burn wounds are MDR, and more than 90% of the isolates are resistant to imipenem and amikacin (21).As can be seen, the amount of resistance in the results of the two mentioned studies is greater than that in our results.
In two different studies, Salehi et al in Tehran and Mihani & Khosravi in Ahvaz reported that more than 70% of P. aeruginosa clinical isolates are resistant to ceftazidime (22,23).In another study, Fallah et al in Tehran reported that 83% of ESBL-producing P. aeruginosa strains isolated from wounds of burnt are resistant to imipenem (24).This rate of resistance in clinical isolates of P. aeruginosa is similar to neighboring countries and African and South American countries, including Pakistan and India, whereas it is significantly higher in North American and European countries (25,26).Differences in the results of various studies can be due to differences in sampling method and sample size.Moreover, differences in geographic area and public health level may be associated with the rate of resistance in P. aeruginosa isolates.
The phenotypic analysis revealed that 65.45% of the P. aeruginosa isolates are ESBL-producing strains which is considered a high ratio compared with other geographic areas in Iran.In three different studies in Iran, Mirsalehian et al, Shahcheraghi et al, and Shakibaie et al have reported that the frequency of ESBL-producing clinical isolates of P. aeruginosa is 40%, 39%, and 34%, respectively (27)(28)(29).The excessive use of broad-spectrum cephalosporins in our province (East Azarbaijan) may be an important reason for the higher frequency of ESBL-producing isolates of P. aeruginosa.Pseudomonas aeruginosa uses numerous mechanisms for the acquisition of drug resistance such as the production of efflux pumps and low membrane permeability.Therefore, the phenotypic identification of ESBL-producing isolates may present false results.In this regard, the molecular analysis of beta-lactamase genes is a precise method for the detection of ESBL-producing isolates of P. aeruginosa.So far, numerous types of beta-lactamase genes have been identified in P. aeruginosa isolates, including VEB, TEM, GES, PER, SHV, CTX, and OXA (3).
Furthermore, the genotypic analysis indicated that the frequency of the blaOXA-4 gene in the detected ESBLproducing isolates of P. aeruginosa is 45.83%.Interestingly, it was found that the presence of the blaOXA-4 gene is significantly associated with resistance to cefepime.In a study in Hamadan, Iran, Sezadehghani et al identified frequency carbapenem encoding genes (OXA), including blaOXA-145 (27.5%), blaOXA-224 (22.0%), blaOXA-539 (20.1%), and blaOXA-675 (11.9%) in clinical isolates of P. aeruginosa, which exhibited MDR (30).In a study by Radmehr et al in North Khorasan, Iran, the frequency of blaOXA-23 gene was reported 61.42% (31).In another study, Bahrami et al reported that the frequency of the blaOXA-48 gene is 12.5% in clinical isolates of P. aeruginosa in Bandar-Abbas, Iran (32).The beta-lactamase encoding genes are transferable between various bacterial strains, which can be a cause of the high prevalence of these genes in clinical isolates of P. aeruginosa in Iranian patients.
In addition to the small sample size of the present study, we only investigated the association between the blaOXA-48 gene and antibiotic resistance of clinical P. aeruginosa.Moreover, we isolated P. aeruginosa strains from a limited type of clinical samples.The investigation of ESBL-producing isolates of P. aeruginosa from other sources such as foods is recommended.
Generally, the present study demonstrated that 65.45% of clinical isolates of P. aeruginosa are phenotypically ESBL producers, 45.83% of which carry the blaOXA-4 gene.Furthermore, we suggest that the blaOXA-4 gene may be associated with the resistance of P. aeruginosa isolates to cefepime antibiotics.Therefore, phenotypically and genotypically detection of ESBL-producing isolates of P. aeruginosa with MDR can be useful in the application of appropriate antibiotics and as a result a better management of hospital infections.

Table 1 .
Antibiotic-Resistant Pattern of Clinical Pseudomonas aeruginosa Isolates

Table 2 .
Frequency of ESBL-Producing Pseudomonas aeruginosa Isolates

Table 3 .
Frequency of the blaOXA-4 Gene in ESBL-Producing Pseudomonas aeruginosa Isolates