Occurrence of class 1, 2, and 3 integrons among multidrug-resistant Pseudomonas aeruginosa in Babylon Province, Iraq

Ahmed Abdulkareem Almuttairi; Anwar A Abdulla

doi:10.4103/MJBL.MJBL_329_22

ORIGINAL ARTICLE

Year : 2023 | Volume : 20 | Issue : 1 | Page : 181-187

Occurrence of class 1, 2, and 3 integrons among multidrug-resistant Pseudomonas aeruginosa in Babylon Province, Iraq

Ahmed Abdulkareem Almuttairi, Anwar A Abdulla
Department of Biology, College of Science, Babylon University, Hillah, Iraq

Date of Submission	14-Dec-2022
Date of Acceptance	11-Jan-2023
Date of Web Publication	29-Apr-2023

Correspondence Address:
Ahmed Abdulkareem Almuttairi
Department of Biology, College of Science, Babylon University, Hillah
Iraq

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/MJBL.MJBL_329_22

Abstract

Background: Clinical management of bacterial infections has faced significant difficulties in recent years due to the advent and spread of multiple drug-resistant (MDR) bacteria. Worldwide, nosocomial infections are brought on by Pseudomonas aeruginosa, a clinically significant Pseudomonas species. Objectives: This research aimed to identify class 1, 2, and 3 integrons in P. aeruginosa in Babylon, Iraq. Materials and Methods: From February 2022 to October 2022, 131 isolates from various sites including (burn, wound, and urine) were collected from different hospitals in Babylon Province for both genders and ages. These isolates were identified using traditional techniques as well as the Vitek 2 system (bioMerieux, France). Pseudomonas aeruginosa isolates were subjected to disc diffusion antimicrobial susceptibility testing. Class 1, 2, and 3 integron-specific primers were used in the polymerase chain reaction technique for the molecular identification of integron genes. Results: Pseudomonas aeruginosa isolates that were 131 (100%) had integron class 1. On the contrary, only five (3.81%) contained a class 2 integron. There was no presence of class 3 integron in any isolate. Conclusion: The MDR P. aeruginosa was highly prevalent (100%) and this suggested that the availability of class 1 integrons in our area was alarmingly high, showing the need for epidemiological monitoring.

Keywords: Antibiotic resistance, integron, Pseudomonas aeruginosa

How to cite this article:
Almuttairi AA, Abdulla AA. Occurrence of class 1, 2, and 3 integrons among multidrug-resistant Pseudomonas aeruginosa in Babylon Province, Iraq. Med J Babylon 2023;20:181-7

How to cite this URL:
Almuttairi AA, Abdulla AA. Occurrence of class 1, 2, and 3 integrons among multidrug-resistant Pseudomonas aeruginosa in Babylon Province, Iraq. Med J Babylon [serial online] 2023 [cited 2023 Jun 11];20:181-7. Available from: https://www.medjbabylon.org/text.asp?2023/20/1/181/375145

Introduction

Pseudomonas aeruginosa frequently infects people with cancer, burn injuries, cystic fibrosis, and other severe illnesses.^[1] Burns are not specific to any given population, age, gender, occupation, or ethnic group; every individual is at risk of any type of burns and in any location.^[2]Pseudomonas aeruginosa infection in burn patients is prevalent and ranks among the most serious life-threatening illnesses in burn units because burn wounds generally serve as a good site for bacterial multiplication due to immune system suppression.^[3] The use of antimicrobial agents is of great importance in the treatment of bacterial infections.^[4]Pseudomonas aeruginosa is typically less resistant to numerous antibiotics by nature as compared with other gram-negative bacilli. Strains of P. aeruginosa that are multiple drug resistant (MDR) are difficult to treat clinically.^[5]

Pseudomonas aeruginosa isolates that are resistant to medication frequently cause burn infections, which can be fatal in medical facilities.^[6] MDR P. aeruginosa outbreaks in healthcare situations are most likely a result of increased antibiotic use. Bacterial resistance is typically explained by the transferable genetic elements’ plasmids, transposons, and integrons or by gene mutation.^[7] Part of antibiotic resistance is due to three different kinds of integrons, known as class 1, class 2, and class 3. They can be recognized by differences in the integrase gene’s intI structure.^[8]

The integron is a genetic component that can play a significant part in the spread of multidrug resistance in gram-negative bacteria, particularly in Pseudomonas.^[9] The integrase genes (intI2 and intI3) are found in class 2 and 3 integrons, and their corresponding produces are 46% and 61% identical to class 1 integrase.^[10] Antimicrobial resistance is attributable to several mechanisms of integrons, beta-lactamases, and efflux pumps.^[11] Integrons have a significant role in the spread of antibiotic resistance; therefore, this study sought to determine the molecular relationships and presence of integrons with multiple drug resistance patterns in P. aeruginosa isolates from medical samples in Babylon Province, Iraq.

Materials and Methods

Study subjects

A cross-sectional study was conducted at various hospitals in Babylon Province from February 2022 to October 2022 using clinical specimens, as listed in [Table 2], and transported to the laboratory for additional examination using common microbiological and biochemical characterization, then confirmed by the Vitek2 system to identified P. aeruginosa isolates.

Table 2: Distribution of Pseudomonas aeruginosa in patients

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Susceptibility testing for antimicrobials

Relative to CLSI, 2022,^[12] antibiotic susceptibility was tested by the disk diffusion test. As a control positive, P. aeruginosa ATCC 27853 (ATCC/USA) was also examined as a reference strain for antibiotic resistance. However, isolates that were multiple-drug resistant were distinct as being resistant to at least three different antibiotic groups.^[13]

Extraction of genomic DNA

Genomic DNA was extracted from P. aeruginosa isolates. The genomic DNA extraction procedure was provided by Favorgen DNA Extraction KIT, Korea and stored at –20°C. DNA concentration and purity of the samples were assessed using Nano-drop (Analytik Jena, Germany).

Polymerase chain reaction assay

The presence of class 1, 2, and 3 integrases was examined in all isolates. For each gene, specific primers were constructed [Table 1]. A final volume of 25 µL was used for the polymerase chain reaction (PCR). Under the following conditions, PCR amplification for the identification of integrase genes intI1 and intI2 was performed as reported,^[14] PCR products were separated on 1% agarose gel, and their expected sizes were checked.^[15]

Table 1: Primers for polymerase chain reaction amplification of integrase genes

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Statistical analysis

Chi-square at a probability of 0.05 was applied to assess the differences of tested variables using statistical package for the social sciences, SPSS version 25.0 (SPSS, IBM, Chicago, Illinois) and the odds ratio was calculated.

Ethical approval

The study was conducted in accordance with the ethical principles that have their origin in the Declaration of Helsinki. It was carried out with patients verbal and written approval before the sample was taken. The study protocol and the subject information and the consent form were reviewed and approved by University of Babylon ethical committee according to document number B220101 (including the number and the date in 17/01/2022) to get this approval.

Results

Isolates

From diverse clinical samples taken from several hospitals in the Babylon Province of Iraq, 131 verified P. aeruginosa isolates were found. Total P. aeruginosa isolates were 51.9 % (68/131) from male and 48.1% (63/131) from female hospitalized patients. The mean (±standard deviation [SD]) age of the group was 34.1 ± 20.6 years. From 385 diverse clinical samples taken from several hospitals in the Babylon province of Iraq, 131 isolates were obtained [Table 2].

Resistances patterns

All 131 P. aeruginosa isolates were tested for susceptibility to 22 antibiotics [Table 3]. The findings suggested that different isolates’ rates of susceptibility to various antibiotics may be observed.

Table 3: Concentrations of antibiotics according to CLSI 2022

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[Figure 1] shows that all P. aeruginosa isolates were resistant to ampicillin, cefixime, and cefotaxime in (100%). The isolates were resistant to Ceftizoxime (99.23%), Cefepime (93.90%), Tetracycline (91.60%), Gentamicin (83.97%), Doxycycline (90.84%), Cefoperazone (84.73%), Tobramycin and Meropenem (81.68%) respectively, Ticarcillin clavulanic (80.91%), Amikacin (79.39 %) Piperacillin (77.86%), Imipenem (74.80%), Levofloxacin (75.57%), Aztreonam (74.05%), Ciprofloxacin (73.28%), Norfloxacin (71.75%), Piperacillin-tazobactam PIT (64.12%), and Doripenem (54.20%). Whereas (40.46%) of the isolates were sensitive to Azithromycin. All isolates (100%) showed MDR resistance to at least three groups of antibiotics, with some isolates displaying resistance to nearly all classes.

Figure 1: Antibiotics susceptibility of P. aeruginosa isolates

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Molecular profiles of intgrons

The PCR result showed 100% isolates, tested positive for the class 1 and 3.82% of isolates tested positive for two integron cassettes [Table 4]. However, none of the isolates had any class 3 integrons [Figure 2][Figure 3][Figure 4].

Table 4: Frequency of integron-positive Pseudomonas aeruginosa isolates based on clinical specimens

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Figure 2: PCR amplification of intI1 gene (457 bp) in P. aeruginosa isolates, on 1.5% agarose at 70 V for 2 h. Positive isolates

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Figure 3: PCR amplification of intI2 gene (789 bp) in P. aeruginosa isolates, on 1.5% agarose at 70 V for 2 h. Lanes (1–5) positive isolates, lane (6) negative isolates. N: negative control

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Figure 4: PCR amplification of intI3 gene (922 bp) in P. aeruginosa isolates, on 1% agarose at 70 V for 2 h. All isolates were negative, N: negative control

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Association of isolates sources, antibiotic resistances with types of integrons

The occurrence of integrons, class 2 and resistance to antibiotics listed in [Table 4] and [Table 5], was found to be significantly correlated.

Table 5: Comparison of the antibiotic resistance between class 2 integron—positive and class 2 integron—negative of Pseudomonas aeruginosa

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Discussion

Pseudomonas aeruginosa-MDR isolates have emerged and spread around the world, raising serious concerns that could make it more difficult to choose empirical agents. It is huge significant pathogens producing a variety of diseases in hospitals and healthcare situations.^[16],[17] Many resistance genes can be found as gene cassettes within integrons, that are found on transmissible plasmids and transposons.^[18] Integrons have been found as a major source of resistance genes and were thought to act as antimicrobial resistance gene reservoirs in microbial communities. Antibiotic resistance mediated by integrons was reported in P. aeruginosa isolates.^[19],[20] Azithromycin had the highest antibiotic susceptibility rate in the current study, with a susceptibility rate of 59.54%, while the majority of P. aeruginosa isolates displayed significant antibacterial agent resistance rates. According to the results of the current study, patients are becoming more resistant (100%) to several categories of antibiotics like ampicillin, cefixime, and cefotaxime. Mutations in the class A -lactamases are the primary source of the resistance to third-generation cephalosporins.^[21] In the middle of the 1990s, the fourth-generation Cephalosporin Cefepime was released into clinical use.^[22] Furthermore, 83.97% and 79.39% of our isolates were resistant to aminoglycoside groups gentamicin and amikacin, respectively. Resistance of P. aeruginosa isolates to aminoglycoside antibiotics in this study was parallel to the study of Poonsuk et al.^[23] In the current work, three classes of integrase genes were screened using PCR amplification, 100% and 3.81% of P. aeruginosa isolates harboring class 1 and 2 integron genes, respectively, but none carried class 3 integron genes among all P. aeruginosa isolates.

Many research has reported on the existence of the intI1 gene in tested isolates of P. aeruginosa.^{[24],[25],[26]} The significance of classes 1 integrons in relative to highly antibiotic-resistant P. aeruginosa isolates was shown in the current investigation and agreed with,^[27] which also highlighted their prevalence and importance. The int2 gene frequency has been reported.^[26],[28] Resistance to several β-lactams, including aminoglycosides, rifamycin, chloramphenicol, and quinolones, is conferred by the class 1 integrons. The attI recombination site, which is recognized by the integrase and serves as a receptor site for incoming gene cassettes, and the intI gene, which codes for a site-specific recombinase, are the two key parts of an integron.^[29] On the basis of the existence of class 1 integrons, the establishment of a high frequency of intI1 in our area raises severe future concerns and has the potential to increase and spread antibiotic resistance. Previous studies found that the intI2 and intI3 genes were absent.^[23],[30] The rise in MDR strains observed in the current investigation may be related to the recent, unrestricted usage of antibiotics in healthcare. The primary cause of MDR, which results in the selection and spread of bacteria resistant to antibiotics in clinical medicine, is the reckless use of antibiotics without antibiotic sensitivity testing.

Conclusions

In this investigation, we found that MDR P. aeruginosa was highly associated with class 1 integrons (100%) suggesting that the frequency of class 1 integrons in our area is alarmingly high, demonstrating the need for epidemiological monitoring.

Acknowledgement

The hospital staff and study participants deserve our sincere gratitude.

Financial support and sponsorship

Nil.

Conflict of interests

There are no conflicts of interest.

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