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ORIGINAL ARTICLE
Year : 2020  |  Volume : 11  |  Issue : 1  |  Page : 19-22  

Antimicrobial resistance and presence of Class 1 integrons in Pseudomonas aeruginosa isolates from burn and wound infections


1 Department of Microbiology, Faculty of Nursing, Bilad Al-Rafidain University College, Baqubah, Iraq
2 Department of Pharmaceutical Sciences, Faculty of Pharmacy, Philadelphia University, Amman, Jordan
3 Department of Biomedical Sciences, College of Clinical Pharmacy, King Faisal University, Al-Ahsa, Saudi Arabia
4 Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, Al-Ahsa, Saudi Arabia

Date of Submission19-May-2020
Date of Decision20-May-2020
Date of Acceptance23-Jun-2020
Date of Web Publication20-Jul-2020

Correspondence Address:
Dr. Qutaiba Ahmed Al Khames Aga
Department of Pharmaceutical Sciences, Faculty of Pharmacy, Philadelphia University, Amman
Jordan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jpnr.JPNR_9_20

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   Abstract 


Objectives: Pseudomonas aeruginosa continues to be the most common pathogen in the nosocomial settings. This organism shows the high level of resistance against various groups of antibiotics. Therefore, this study investigates the association between the Class 1 integron and multidrug-resistant P. aeruginosa bacterium isolated from burn and wound infections. Materials and Methods: A total of 184 isolates of P. aeruginosa were collected from nonrepetitive burn and wound samples (n = 866). Kirby–Bauer disk-diffusion test, a standard technique, was used to perform antibiotic susceptibility testing. Class 1 integron genes in P. aeruginosa were identified using the polymerase chain reaction. Results: The antibiotic resistance rate ranged between 19–100% for 184 isolates of P. aeruginosa tested. Polymerase chain reaction amplification revealed that 46.73% (86/184) of P. aeruginosa strains carried Class 1 integron gene, that strains also showed resistance to majority of the antibiotics tested. Negatively, most of the Class 1 integron genes were isolated from wound infections (n = 45) than burn infections (n = 41). Conclusion: The current study revealed that P. aeruginosa isolates that carrying Class 1 integron possess higher level of resistance to amoxicillin, ceftazidime, aztreonam, meropenem, tobramycin, piperacillin, gentamicin, ciprofloxacin, and netilmicin. Therefore, it is essential to conduct antibiotic surveillance and molecular epidemiological studies to practice effective empirical therapies across the hospital settings.

Keywords: Antibiotics, infections, integron, multidrug resistant


How to cite this article:
Hammadi AH, Aga QA, Nimer NA, Shinu P, Nair AB. Antimicrobial resistance and presence of Class 1 integrons in Pseudomonas aeruginosa isolates from burn and wound infections. J Pharm Negative Results 2020;11:19-22

How to cite this URL:
Hammadi AH, Aga QA, Nimer NA, Shinu P, Nair AB. Antimicrobial resistance and presence of Class 1 integrons in Pseudomonas aeruginosa isolates from burn and wound infections. J Pharm Negative Results [serial online] 2020 [cited 2020 Aug 11];11:19-22. Available from: http://www.pnrjournal.com/text.asp?2020/11/1/19/290212




   Introduction Top


Pseudomonas aeruginosa causes frequent infections in patients with burn injuries, cystic fibrosis, cancer, and other debilitating diseases.[1] Burn infections are frequently caused by drug resistant strains P. aeruginosa and that accounts to be a life-threatening conditions in hospital settings.[2] Infections due to multidrug-resistant strains of P. aeruginosa are usually associated with substantial mortality rates, particularly among patients with burn injuries.[3]P. aeruginosa withstand adverse environmental conditions (such as disinfectant-mediated killing) and also possess various intrinsic antibiotic resistance mechanisms and thereby makes it difficult to eliminate from the hospital settings. This type of intrinsic antibiotic resistance mechanisms includes: low outer membrane permeability, enzymatic modification of antibiotics (for instance; β-lactamase production) over expression of efflux pump systems, chromosomal mutations or may be via the acquirement of drug-resistant genes located on plasmids or transposons. Most of the resistance genes are collectively located as separate small mobile units in the core bacterial genome known as integrons.[4],[5] Integrons are genetic elements which are responsible for coding and transfer of drug-resistant determinants.[6] The spread of these drug-resistant genes (may be via horizontal transfer) might have resulted in the development of antibiotic resistance among various bacterial species.[7]

Various studies have been conducted to investigate the incidence of multidrug-resistant mechanisms prevailing in Gram-negative bacterial species (for instance P. aeruginosa) across the globe.[8],[9],[10],[11],[12],[13] However, high mortality rate associated with P. aeruginosa infections (particularly among patients with burn injuries), wide distribution of drug-resistant strains of P. aeruginosa in the hospital settings, lack of data about the distribution of molecular determinants of multidrug-resistant P. aeruginisa in Iraq, encouraged us to design the current study. Considering these parameters, the current study was intended to ascertain the association between the Class 1 integron and the multidrug-resistant P. aeruginosa. In this context, this study investigates the possible relation between Class 1 integron gene and the antibiotic resistance shown by P. aeruginosa. Bacterial isolation was carried out from the samples of patients infected with burn and wound. Samples were cultured and identified by standard laboratory protocol. Antimicrobial susceptibility testing was conducted against antimicrobial agents. Class 1 integrons genes were identified using the polymerase chain reaction amplification method.


   Materials and Methods Top


Study setting and participants

This prospective study was conducted between September 2018 and January 2019, which included 866 clinical samples that collected from patients with burn (n = 345) and wound (n = 521) infections. Both male and female patients (male/female ratio 1.17) with age ranged between 20 and 40 years (standard deviation ± 5.36) who attended Al Numan Teaching Hospital, Educational Laboratories in Medical City Hospital and Burn Hospital in Medical City, Diyala were incorporated in the study.

Laboratory tests

Isolation of bacteria

All the clinical samples were inoculated on MacConkey, cetrimide and blood agar. The isolates were recognized using the standard laboratory protocol (more specifically; API20E system [Biomerieux Inc., Durham, United States] for the identification of P. aeruginosa).

Antibiotic susceptibility testing

Antibiotic susceptibility testing was carried out on Mueller–Hinton agar (Himedia, Mumbai, India) against most commonly used antibiotics (Turkey, Bioanalyse) as described by the Kirby-Bauer disc-diffusion method. The antibiotics used for testing include: Amoxicillin (25 mg/ml), ceftazidime (30 mg/ml), aztreonam (30 mg/ml), ciprofloxacin (10 mg/ml), gentamicin (10 mg/ml), imipenem (10 mg/ml), meropenem (10 mg/ml), netilmicin (30 mg/ml), piperacillin (100 mg/ml), and tobramycin (10 mg/ml). The interpretation of antibiotic microbial testing was carried out as per the Clinical Laboratory Standards Institute.[14]P. aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) were utilized as quality control strains for antibiotic susceptibility testing.

Molecular analysis

DNA extraction and polymerase chain reaction

The DNA of P. aeruginosa isolates was extracted using a DNA extraction kit (G-spin, iNtRON Biotechnology, Sangdaewon-Dong, South Korea) and manufactures instructions were strictly followed while performing the procedure. Using primers listed in the [Table 1], integron gene detection was accomplished through the polymerase chain reaction amplification technique. The reaction mixture was prepared using 5 U/μl from Taq Polymerase chain reaction PreMix, forward primer (10 μM), reverse primer (10 μM), DNA extract (1.5 μl), and the volume was made to 25 μl by mixing double distilled water, the mixture is then placed in the mixer device, and the polymerase chain reaction (Bioneer, Daejeon, Korea) was performed as per the conditions described in the [Table 2].
Table 1: Forward and reverse primer

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Table 2: Polymerase chain reaction methods and conditions

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Statistical analysis

The study groups were compared against all the variables including the frequency of occurrence of introns. Independent sample Chi-square test used for analyzing the categorical variables of integron positive and negative strains in relation to resistance. P < 0.05 was treated as significant in all statistical data analyzed using the SPSS software version 17.9 (SPSS, Chicago, IL, United States).

Ethics statement

Research Ethics Committee in Bilad Alrafidain University, Diyala and Al Numan Teaching Hospital Committee approved the study protocol (BRUC/2376/23/08/2018).


   Results Top


[Table 1] and [Table 2] shows primers and polymerase chain reaction conditions used in detecting Class 1 integron genes in P. aeruginosa. The distribution of P. aeruginosa isolates obtained from wound and burn infections is summarized in [Table 3]. The current study analyzed 184 isolates of P. aeruginosa strains that obtained from burn 39.67% (73/184) and wound 60.32% (111/184) infections. It is evident from [Table 3] that 52.32% (45/86) of Class 1 integron-positive isolates were obtained from wound infections. [Table 4] illustrates the association of Class 1 integron gene and antibiotic susceptibility pattern. It is apparent from [Table 4] that all the strains that tested were resistant to amoxicillin 100% (184/184), ceftazidime 100% (184/184) followed by ciprofloxacin 86.41% (159/184), piperacillin 81.52% (150/184), gentamicin 84.24% (155/184), meropenem 76.08% (140/184), netilmicin 73.91% (136/184), tobramycin 73.36% (135/184), aztreonam 63% (116/184), and to a lesser extent for imipenem 19.56% (36/184), respectively.
Table 3: Distribution of Pseudomonas aeruginosa isolates obtained from wound and burn infections

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Table 4: Association between the presence of class 1 integron gene and antibiotic resistance

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   Discussion Top


P. aeruginosa is an opportunistic pathogen that cause severe infections in diabetes, wound, and burn patients, particularly caused by multidrug-resistant strains of P. aeruginosa.[3] The major mechanism of development of multidrug resistance in Gram-negative bacterial isolates include the horizontal gene transfer, production of various enzymes, and efflux pump mechanisms. Further, in the process of horizontal gene transfer, bacteria exchange various mobile genetic elements such as plasmids, transposons, and integrons between different species of Gram-negative bacteria.[1],[3] This study investigates the incidence of antimicrobial resistance and presence of Class 1 integrons in P. aeruginosa isolates obtained from burn and wound infections. Of the 184 isolates of P. aeruginosa strains analyzed, majority of the Class1 integron genes were detected from wound infections rather than burn infections. However, studies conducted across the world shows the wide spread dissemination of Class 1 integron genes in various Gram-negative isolates that obtained from burn infections.[9] This higher distribution of Class 1 integron genes in wound specimens may indicate the community distribution of these genes, more specifically; most of the wound samples that were obtained from out-patients. The low level of antimicrobial resistance against imipenem observed (19.56%) in this study, which is also in accordance with earlier reports where it ranged 3.77–17.86% against P. aeruginosa isolates.[15],[16] Azimi et al.[17] reported that P. aeruginosa isolates showed resistance to 40%–43% to all tested drugs in contrast to the current study in which the resistance pattern varied between 63% and 100% (with an exception of imipenem). This variation in the bacterial resistance pattern may depend on many factors such as geographic variability, infection control measures practiced, antibiotic policies followed in the hospital settings.[18]

Antibiotic susceptibility testing results of P. aeruginosa demonstrates that most of the isolates that carrying Class 1 integron gene demonstrated resistance against majority of the antibiotics tested [Table 4]. It is evident from [Table 4] that P. aeruginosa strains that possess Class 1 integron gene showed higher level of resistance against amoxicillin, ceftazidime, meropenem, tobramycin, piperacillin, gentamycin, ciprofloxacin, and netilmicin. However, a low level of antibiotic resistance was noted among P. aeruginosa (Class 1 integron negative) strains. This variation in the antibiotic susceptibility pattern between Class 1 integron positive and negative strains may be due to the transposition. Transposition is a gene transfer mechanism that helps in the movement of integrons genes between the plasmids of different species of bacteria. More specifically; these integrons are mobile genetic elements located in the genome of P. aeruginosa and it may capture external drug resistance gene cassettes to enhance multidrug resistance.[19]

Literature indicate the distribution of Class 1 integron genes in different bacterial species, particularly among Gram-negative bacterial isolates.[20] The frequency of distribution of Class 1 integron gene in this study was found to be 46.73% (86/184) among the total P. aeruginosa strains isolated. This distribution is comparable with earlier studies that conducted across the world wherein the prevalence of Class 1 integron gene varied between 38% and 41%.[20],[21] However, this is the first such kind of study that investigated the incidence of Class 1 integron gene in this geographical area. Further, the difference in the rate of distribution of Class 1 integron genes may be attributed to the inclusion of various types of clinical samples more specifically; other studies used urine, blood and sputum samples for the detection of Class 1 integron gene.[20],[21],[22] The other possible reasons for this differences in the gene carriage of P. aeruginosa isolates may be attributed to the strains of study population, organism isolated, antibiotic usage, and infection prevention practices.[23] Further, the data in this study demonstrated significant correlation between bacterial strains that carrying Class 1 integron gene and antibiotic susceptibility pattern with relatively higher sensitivity to imipenem.


   Conclusion Top


This study revealed that P. aeruginosa isolates that carrying Class 1 integron possess higher level of resistance to amoxicillin, ceftazidime, aztreonam, meropenem, tobramycin, piperacillin, gentamicin, ciprofloxacin, and netilmicin. However, it possesses low level of resistance against imipenem. Therefore, it is essential to conduct antibiotic surveillance and molecular epidemiological studies to practice effective empirical therapies across the hospital settings.

Acknowledgment

The authors would like to thank Al Numan Teaching Hospital, Educational Laboratories in Medical City Hospital and Burn Hospital in Medical City, Diyala for their support in completing this study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest



 
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Chen J, Su Z, Liu Y, Wang S, Dai X, Li Y, et al. Identification and characterization of Class 1 integrons among Pseudomonas aeruginosa isolates from patients in Zhenjiang, China. Int J Infect Dis 2009;13:717-21.  Back to cited text no. 21
    
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Mobaraki S, Aghazadeh M, Soroush Barhaghi MH, Yousef Memar M, Goli HR, Gholizadeh P, et al. Prevalence of integrons 1, 2, 3 associated with antibiotic resistance in Pseudomonas aeruginosa isolates from Northwest of Iran. Biomedicine (Taipei) 2018;8:2.  Back to cited text no. 22
    
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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