Original Article

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

Please cite this article as follows: Gholampour Matin M, Shapouri R, Nahaei M, Mohammadi Roknabadi M, Shokri R. Genotypic investigation of antibiotic resistant blaOXA-4 gene in clinical isolates of Pseudomonas aeruginosa. Avicenna J Clin Microbiol Infect. 2023; 10(3):95-99. doi:10.34172/ajcmi.3471

Introduction

Pseudomonas aeruginosa is the most pathogenic member of the Pseudomonadaceae family that includes gram-negative, non-fermenting, obligatory aerobic, oxidase-positive 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 beta-lactam antibiotics via hydrolysis central core of the beta-lactam 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.

Materials and Methods

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, H₂S 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.

Antibiotic Resistance Analysis

The antimicrobial-resistant pattern of the approved isolates was evaluated by the disk diffusion method as described by the Clinical and Laboratory Standards Institute. We used numerous antibiotic discs (Padtanteb, Iran), including gentamicin (10 µg), tobramycin (5 µg), ciprofloxacin (5 µg), amikacin (30 µg), ceftazidime (30 µg), piperacillin (100 µg), imipenem (30 µg), and cefepime (30 µg). Furthermore, P. aeruginosa ATCC 27853 was considered as the control strain (5).

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

The combination disc method was applied for phenotypic detection of ESBL β-lactamases by cefotaxime-clavulanic acid (30 μg-10 μg), ceftazidime-clavulanic acid (30 µg-10 µg), cefotaxime (30 µg), and ceftazidime (30 µg) antibiotic discs (Padtanteb, Iran). The isolates with simultaneous resistance against at least 3 antibiotics were considered multidrug-resistant (MDR), while the isolates with ≥ 5 mm growth inhibition halo by combined discs were considered ESBL-positive as compared to growth inhibition halo by alone discs (6).

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.

Results

Bacterial Isolates

We collected 110 clinical isolates of P. aeruginosa out of 622 specimens identified by biochemical analysis. The clinical isolates of P. aeruginosa included 34 cases (30.90%) from wound samples, 26 cases (23.65%) from tracheal aspirate samples, 27 cases (24.55%) from urine samples, and 23 cases (20.90%) from blood samples.

Antibiotic Resistance Pattern

The results of the disk diffusion method demonstrated that P. aeruginosa isolates are most sensitive to amikacin (65.45%) antibiotic and most resistant to ceftazidime (86.36%), ciprofloxacin (80.00%), and tobramycin (76.36%) antibiotics (Table 1).

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 ESBL-positive 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 of 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 (27-29). 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 ESBL-producing 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.

Acknowledgments

This article was extracted from the Ph.D. project of MGM which was supervised by RS and MN and advised by MMR and RS.

Authors’ Contribution

Conceptualization: Reza Shapouri.

Data Collection: Milad Gholampour Matin.

Formal Analysis: Reza Shapouri.

Funding Acquisition: Milad Gholampour Matin.

Investigation: Milad Gholampour Matin.

Methodology: Milad Gholampour Matin.

Project administration: Reza Shapouri, Mohammadreza Nahaei.

Software: Mojtaba Mohammadi Roknabadi.

Supervision: Reza Shapouri, Mohammadreza Nahaei.

Validation: Rasoul Shokri, Mohammadreza Nahaei.

Writing–original draft: Milad Gholampour Matin, Mojtaba Mohammadi Roknabadi.

Competing Interests

There is no conflict of interests as stated by the authors.

Ethical Approval

Not applicable.

References

1. Rudra B, Gupta RS. Phylogenomic and comparative genomic analyses of species of the family Pseudomonadaceae: proposals for the genera Halopseudomonas gen nov and Atopomonas gen nov, merger of the genus Oblitimonas with the genus Thiopseudomonas, and transfer of some misclassified species of the genus Pseudomonas into other genera. Int J Syst Evol Microbiol 2021; 71(9):005011. doi: 10.1099/ijsem.0.005011 [Crossref] [ Google Scholar]
2. Soleymanzadeh Moghadam S, Khodaii Z, Fathi Zadeh S, Ghooshchian M, Fagheei Aghmiyuni Z, Mousavi Shabestari T. Synergistic or antagonistic effects of probiotics and antibiotics- alone or in combination- on antimicrobial-resistant Pseudomonas aeruginosa isolated from burn wounds. Arch Clin Infect Dis 2018; 13(3):e63121. doi: 10.5812/archcid.63121 [Crossref] [ Google Scholar]
3. Pang Z, Raudonis R, Glick BR, Lin TJ, Cheng Z. Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies. Biotechnol Adv 2019; 37(1):177-92. doi: 10.1016/j.biotechadv.2018.11.013 [Crossref] [ Google Scholar]
4. Curran CS, Bolig T, Torabi-Parizi P. Mechanisms and targeted therapies for Pseudomonas aeruginosa lung infection. Am J Respir Crit Care Med 2018; 197(6):708-27. doi: 10.1164/rccm.201705-1043SO [Crossref] [ Google Scholar]
5. Rahimzadeh Torabi L, Doudi M, Golshani Z. The frequency of blaIMP and blaVIM carbapenemase genes in clinical Isolates of Pseudomonas aeruginosa in Isfahan medical centers. Med J Mashhad Univ Med Sci 2016; 59(3):139-47. doi: 10.22038/mjms.2016.7714.[Persian] [Crossref] [ Google Scholar]
6. Tavajjohi Z, Moniri R, Khoeshidi A. Frequency of extended-spectrum beta-lactamase (ESBL) multidrug-resistance produced by Pseudomonas aeruginosa isolated from clinical and environmental specimens in Kashan Shahid Beheshti hospital during 2010-11. Feyz 2011;15(2):139-45. [Persian].
7. Taghinejad J, Hosseinzadeh M, Molayi Kohneshahri S, Javan Jasor V. Pseudomonas aeruginosa: a biological review. Lab Diagn 2017;8(34):67-82. [Persian].
8. Drawz SM, Bonomo RA. Three decades of beta-lactamase inhibitors. Clin Microbiol Rev 2010; 23(1):160-201. doi: 10.1128/cmr.00037-09 [Crossref] [ Google Scholar]
9. Nzouankeu A, Fonkoua MC, Wouafo MW, Njine TN, Aidara-Kane AA, Ngandjio A. Molecular characterization of multidrug resistant Salmonella from chicken and human in Yaounde. Med Res Arch 2016; 4(8):1-29. [ Google Scholar]
10. Hashemi A, Fallah F, Taherpour A, Goudarzi H, Tarashi S, Erfanimanesh S, et al. Detection of metallo-beta-lactamases, extended-spectrum beta-lactamases (ESBLs), outer membrane porins among Klebsiella pneumoniae strains isolated from hospitalized patients in Tehran. J Adv Med Biomed Res 2015;23(98):89-102. [Persian].
11. Ur Rahman S, Ali T, Ali I, Khan NA, Han B, Gao J. The growing genetic and functional diversity of extended spectrum beta-lactamases. Biomed Res Int 2018; 2018:9519718. doi: 10.1155/2018/9519718 [Crossref] [ Google Scholar]
12. Franco MR, Caiaffa-Filho HH, Burattini MN, Rossi F. Metallo-beta-lactamases among imipenem-resistant Pseudomonas aeruginosa in a Brazilian university hospital. Clinics (Sao Paulo) 2010; 65(9):825-9. doi: 10.1590/s1807-59322010000900002 [Crossref] [ Google Scholar]
13. Abaza AF, El Shazly SA, Selim HSA, Aly GSA. Metallo-beta-lactamase producing Pseudomonas aeruginosa in a healthcare setting in Alexandria, Egypt. Pol J Microbiol 2017; 66(3):297-308. doi: 10.5604/01.3001.0010.4855 [Crossref] [ Google Scholar]
14. Mahdavi S, Isazadeh A. Lactobacillus casei suppresses hfq gene expression in Escherichia coli O157:H7. Br J Biomed Sci 2019; 76(2):92-4. doi: 10.1080/09674845.2019.1567903 [Crossref] [ Google Scholar]
15. Mahdavi S, Tanhaeivash E, Isazadeh A. Investigating the presence and expression of stx1 gene in Escherichia coli isolated from women with urinary tract infection using real-time PCR in Tabriz, Iran. Int J Enteric Pathog 2018; 6(4):104-7. doi: 10.15171/ijep.2018.26 [Crossref] [ Google Scholar]
16. Opazo A, Domínguez M, Bello H, Amyes SG, González-Rocha G. OXA-type carbapenemases in Acinetobacter baumannii in South America. J Infect Dev Ctries 2012; 6(4):311-6. doi: 10.3855/jidc.2310 [Crossref] [ Google Scholar]
17. Rawat D, Nair D. Extended-spectrum β-lactamases in gram-negative bacteria. J Glob Infect Dis 2010; 2(3):263-74. doi: 10.4103/0974-777x.68531 [Crossref] [ Google Scholar]
18. Siddique A, Azim S, Ali A, Andleeb S, Ahsan A, Imran M. Antimicrobial resistance profiling of biofilm forming non typhoidal Salmonella enterica isolates from poultry and its associated food products from Pakistan. Antibiotics (Basel) 2021; 10(7):785. doi: 10.3390/antibiotics10070785 [Crossref] [ Google Scholar]
19. Yari Z, Mahdavi S, Khayati S, Ghorbani R, Isazadeh A. Evaluation of antibiotic resistance patterns in Staphylococcus aureus isolates collected from urinary tract infections in women referred to Shahid Beheshti educational and therapeutic center in Maragheh city, year 2016. Med J Tabriz Uni Med Sci Health Serv 2020; 41(6):106-12. doi: 10.34172/mj.2020.013.[Persian] [Crossref] [ Google Scholar]
20. Fazeli H, Moslehi Z, Irajian G, Salehi M. Determination of drug resistance patterns and detection of bla-VIM gene in Pseudomonas aeruginosa strains isolated from burned patients in the Emam Mosa Kazem hospital, Esfahan, Iran (2008-9). Iran J Med Microbiol 2010;3(4):1-8. [Persian].
21. Ranjbar R, Owlia P, Saderi H, Mansouri S, Jonaidi-Jafari N, Izadi M. Characterization of Pseudomonas aeruginosa strains isolated from burned patients hospitalized in a major burn center in Tehran, Iran. Acta Med Iran 2011; 49(10):675-9. [ Google Scholar]
22. Salehi M, Hekmatdoost M, Hosseini F. Quinolone resistance associated with efllux pumps mexAB-oprM in clinical isolates of Pseudomonas aeruginosa. J Microbial World 2014;6(4):290-8. [Persian].
23. Mihani F, Khosravi A. Isolation of Pseudomonas aeruginosa strains producing metallo beta lactamases from infections in burned patients and identification of blaIMP and blaVIM genes by PCR. Iran J Med Microbiol 2007;1(1):23-31. [Persian].
24. Fallah F, Shams Borhan R, Gholinejad Z, Zahirnia Z, Adabiyan S, Sattarzadeh Tabrizi M, et al. Detection of blaIMP and blaVIM metallo-beta-lactamases genes in Pseudomonas aeruginosa strains isolated from wound of burnt patients in Tehran Shahid Motahari hospital during 2011, Iran. Qom Univ Med Sci J 2013;7(5):21-7. [Persian].
25. Hammami S, Gautier V, Ghozzi R, Da Costa A, Ben-Redjeb S, Arlet G. Diversity in VIM-2-encoding class 1 integrons and occasional blaSHV2a carriage in isolates of a persistent, multidrug-resistant Pseudomonas aeruginosa clone from Tunis. Clin Microbiol Infect 2010; 16(2):189-93. doi: 10.1111/j.1469-0691.2009.03023.x [Crossref] [ Google Scholar]
26. Nagaveni S, Rajeshwari H, Oli AK, Patil SA, Chandrakanth RK. Widespread emergence of multidrug resistant Pseudomonas aeruginosa isolated from CSF samples. Indian J Microbiol 2011; 51(1):2-7. doi: 10.1007/s12088-011-0062-0 [Crossref] [ Google Scholar]
27. Mirsalehian A, Feizabadi M, Akbari Nakhjavani F, Jabal Ameli F, Goli H. Prevalence of extended spectrum beta lactamases among strains of Pseudomonas aeruginosa isolated from burn patients Tehran Univ Med J 2008;66(5):333-7. [Persian].
28. Shahcheraghi F, Nasiri S, Noveiri H. The survey of genes encoding beta-lactamases, in Escherichia coli resistant to beta-lactam and non-beta-lactam antibiotics. Iran J Basic Med Sci 2010; 13(1):230-7. doi: 10.22038/ijbms.2010.5068 [Crossref] [ Google Scholar]
29. Shakibaie MR, Shahcheraghi F, Hashemi A, Saeed Adeli N. Detection of TEM, SHV and PER type extended-spectrum ß-lactamase genes among clinical strains of Pseudomonas aeruginosa isolated from burnt patients at Shafa-hospital, Kerman, Iran. Iran J Basic Med Sci 2008; 11(2):104-11. doi: 10.22038/ijbms.2008.5220 [Crossref] [ Google Scholar]
30. Sezadehghani A, Dehbashi S, Tahmasebi H, Arabestani MR. Detection of blaOXA-145, blaOXA-224, blaOXA-539, and blaOXA-675 genes and carbapenem-hydrolyzing class D β-lactamases (CHDLs) in clinical isolates of Pseudomonas aeruginosa collected from west of Iran, Hamadan. Int J Microbiol 2022; 2022:3841161. doi: 10.1155/2022/3841161 [Crossref] [ Google Scholar]
31. Radmehr M, Moghbeli M, Ghasemzadeh Moghadam H. Determination of antibiotic susceptibility pattern of Pseudomonas aeruginosa and the prevalence of blaOXA-23 gene in isolates from a hospital in North Khorasan. J Microbial World 2021; 14(4):59-68. doi: 10.30495/jmw.2021.690460.[Persian] [Crossref] [ Google Scholar]
32. Bahrami M, Mohammadi-Sichani M, Karbasizadeh V. Prevalence of SHV, TEM, CTX-M and OXA-48 β-lactamase genes in clinical isolates of Pseudomonas aeruginosa in Bandar-Abbas, Iran. Avicenna J Clin Microbiol Infect 2018; 5(4):86-90. doi: 10.34172/ajcmi.2018.18 [Crossref] [ Google Scholar]