Investigation of antimicrobial susceptibility, class I and II integrons among Pseudomonas aeruginosa isolates from hospitalized patients in Isfahan, Iran

Objectives The role of integrons in the transfer of antibiotic resistance is one of the important issues, therefore, this study is aimed to investigate antibiotic resistance pattern and prevalence of class 1 and 2 integrons in P. aeruginosa isolated. Results Out of 72 confirmed P. aeruginosa isolates, 50% were from ICU patients. Antibacterial susceptibility pattern showed that isolates were most resistant to ceftazidime (76.4%) and colistin was the most effective antibiotic (100%) and molecular analysis of class I and II integrons showed 55.5% and 29.1% of isolates were positive, respectively and the proportions of MDR isolates were significantly higher among integron-positive isolates with 73.6% compared to negative isolates with 22.9%. Our results showed that there was a correlation among class 1 and 2 integrons with MDR P. aeruginosa isolates. According to the importance of integrons in acquisition and dissemination of antibiotics resistance genes, the performance of antibiotic surveillance programs and investigating the role of integrons is recommended to control the spreading of antibiotics resistance genes.


Introduction
Pseudomonas aeruginosa is a non-fermenting aerobic Gram-negative bacillus known as a significant pathogen leading to severe infections in hospitals and rarely causes infection in the natural host [1]. P. aeruginosa is an important opportunistic pathogen leading to severe infections in patients with cystic fibrosis, neutropenia, iatrogenic immunosuppression, or severe burns [2,3]. The increasing use of antibiotics is likely the main reason for the evolution of multidrug-resistant (MDR) P. aeruginosa outbreaks in clinical settings [4,5]. Generally, bacterial resistance can be explained by either the mutation of genes or exchangeable genetic elements such as plasmids, transposons, and integrons [6]. Integrons are transportable genetic elements which can transfer the antibiotic resistance genes. These elements can be placed in different parts of plasmids and chromosomes. Integrons are able to capture external drug resistance gene cassettes and incorporate them using site-specific recombination. It is potentially a major agent in the rapid spread of resistance in bacteria, especially among the Gram-negative [6]. The overall structure of integrons includes an integrase gene, two stable conservative reigns called sul1 and int1 as well as a variable region of gene cassettes between the two conserved segments [7]. According to the integrase gene, four classes of integrons have been described in Gram-negative organisms, however, class 1 integrons have been described to be the most prevalent in clinical isolates, carrying single or several gene cassettes, which confer resistance to a wide range of antibiotics such as β-lactams, fluoroquinolones, aminoglycosides,  [7]. The second class of integrons (class 2) is commonly found to be associated with the Tn7 transposon family and it contains integrase gene (intI2), one recombinant location (aatI 2) and a cassette gene without SulI in its 3′ terminal region. Class 2 integrons have been reported in some species of Gram-negative bacteria such as Acinetobacter, Enterobacteriaceae, Salmonella and Pseudomonas [8]. Compared with class 2 integron, class 3 integron contains a similar structure and this class of integrin have been described as rare and found only within a few microorganisms such as Serratia marcescens, Klebsiella pneumoniae and P. aeruginosa [8][9][10]. Furthermore, class 4 integron which harbor a wide range of gene cassettes encoding antibiotic resistance and pathogenicity was firstly identified on the small chromosome of Vibrio cholerae [8].

BMC Research Notes
Regarding the significant role of P. aeruginosa in nosocomial infections and also the role of integrons in the dissemination of antibiotic resistance, this study is aimed to evaluate the frequency of class 1 and 2 integrons in P. aeruginosa isolates of nosocomial infection in order to determine the antibiotic resistance pattern of this bacterium.

Methods
A cross-sectional study was performed during the period April 2016 to August 2016 at a teaching hospital affiliated to Isfahan University of Medical Sciences, Isfahan, Iran. The non-replicated P. aeruginosa isolates were obtained from various clinical specimens such as blood, wounds, urine and other samples and transported to the laboratory for further analysis. The specimens were cultured on blood agar and MacConkey's agar (Merck, Germany) and incubated overnight at 37 °C. Bacterial isolates were identified as P. aeruginosa using standard microbiological methods, including Gram staining, production of acid from different sugars, catalase, oxidase, motility, and genotypic method (the presence of the tox-A gene). The confirmed isolates were stored at − 80 °C in brain heart infusion (BHI) broth containing 20% glycerol [11,12].
A simple boiling method was used to extract genomic DNA from P. aeruginosa isolates as described previously [14]. The polymerase chain reaction (PCR) was carried out to detect the tox-A gene and the presence of class 1 and 2 integrons cassette using the specific primers [15].
PCR amplification consisted of a pre-denaturation step at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 45 s, primer annealing at 54 °C for intI1 and intI2 and extension at 72 °C for 60 s, and a final extension at 72 °C for 7 min. The PCR products were analyzed on agarose gel with KBC power load dye (CinnaGen Co. Iran) and finally visualized in gel documentation system. Positive results were confirmed by direct sequencing of the PCR products.
SPSS Statistics (IBM Corp. Released 2012. IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp.) was used for statistical analysis. Chi square or Fisher's exact test was used to determine any statistical association. Statistical significance was regarded as p-values < 0.05. The results are showed using descriptive statistics in terms of relative frequency.

Results
A total of 72 confirmed P. aeruginosa isolates were obtained from various clinical samples of a selected teaching hospital in Isfahan, Iran. Totally, 66.7% (48/72) P. aeruginosa isolates were obtained from male and 33.3% (24/72) from female hospitalized patients. The distribution of P. aeruginosa isolates in different hospital wards and clinical specimens showed that half of the isolates were collected from ICU and most of the isolates were isolated from trachea samples. Table 1 shows the distribution of clinical specimens and different wards based on integron-positive P. aeruginosa isolates.
Antibacterial susceptibility pattern showed a high rate of antibiotic resistance to ceftazidime 76.4% followed by ciprofloxacin 73.6%. However, all of the isolates were susceptible to colistin (100%) and the lowest antibiotic resistance rates were seen against amikacin (41.6%) followed by piperacillin/tazobactam (48.6%).
PCR amplification of the two class 1 and 2 integrons cassette showed that 55.5% and 29.1% of isolates were positive for class 1 and 2 integrons, respectively.
The high level of resistance in class 1 and 2 integronpositive isolates were seen to ceftazidime (82.5) and ciprofloxacin (76.2%), respectively, whereas amikacin and piperacillin/tazobactam were the most effective antibiotics to integron-positive isolates. The full antibiotic resistance pattern of the integron-positive and integronnegative isolates are presented in Tables 2 and 3. According to our results, a significant correlation between the presences of class I integron and a higher rate of resistance related to meropenem was seen (p-value < 0.001).

Discussion
Pseudomonas aeruginosa is one of the most important pathogen causing a wide range of infections in the hospitals and healthcare settings [16]. The spread of resistance to antimicrobial agents and the emergence of clinical multidrug resistance P. aeruginosa isolates in clinical settings has become a serious problem in the treatment of nosocomial infections worldwide [17,18]. Resistance to antimicrobial agents can be originated from many resistance genes that present as gene cassettes and transferred through integrons which are located on transmissible plasmids, the chromosome, and transposons [10]. In the present study, based on antibiotic susceptibility pattern, colistin was the most effective antibiotic agent with the susceptibility rate of 100%, while most of P. aeruginosa isolates showed high resistance rate to antimicrobial agents. In accordance to our results, Khosravi [19][20][21].
In another study carried out by Fazeli et al. [22] in our region, lower resistance rate was described for quinolones and cephems agents. Among them, a resistance rate of 63% and 63.1% were reported for both ciprofloxacin and ceftazidime, respectively. Whereas, our results showed a higher resistance to quinolones and cephems groups especially ciprofloxacin and ceftazidime which demonstrated an increasing trend of this resistance in hospitalized patients in Isfahan. In our finding, the rate   Colistin 0 0 -of resistance to amikacin was 41% that was lower than reports from the south of Iran and were also reported in other parts of the world [19,[21][22][23][24]. To our knowledge, this is the first study addressing the investigation of a different class of integron in P. aeruginosa isolates in our region, therefore, this present study demonstrated the importance and frequency of classes 1 and 2 integrons and the importance of them in relation to high antibiotic resistance P. aeruginosa isolates.
In the present study, 55.5% and 29% of P. aeruginosa isolates were contained class 1 and 2 integrons, meanwhile, 16.6% of isolates carried both classes of integron genes, simultaneously.
The overall data show that the prevalence of int1 gene in clinical isolates was closest to most of the previous investigations from Iranian studies ranging from 27.5 to 66% and the results of studies conducted in other countries [20,21,[25][26][27][28].
The emergence of the high frequency of intI1 in our region may explain a serious concern in the future and can cause an increase and dissemination of antibiotic resistance that is related to the presence of class 1 integron. In similar studies from Iran, due to inappropriate use of antibiotics, the origin of infections and geographical distribution, the prevalence of class 2 gene in clinical isolates of P. aeruginosa is variable. Accordingly, in contrast to our finding, other studies have reported a variable frequency of class 2 integrons among clinical P. aeruginosa. Khosravi [19,21,29].
According to the previous finding, there is an association between the presence of multiple resistance and different classes of Integron, especially class 1 integrons [30]. Bearing in mind our results, in compared to negative isolates, the proportion of MDR isolates among integron-positive isolates were considerably higher, which can confirm the importance of these elements in the spreading of resistance genes among pathogens.
In agreement with our findings, a high rate of MDR isolates among integron-positive were reported by Ebrahimpour et al. [31]. Moreover, Khosravi et al. [19] also showed there is a correlation between the presence of class 1 integrons and MDR isolate and high antimicrobial resistance especially for gentamicin (94.62%), ciprofloxacin (93.54%), and meropenem (90.32%).
Although the resistance rate of meropenem was significantly differenced in class 1 integron-positive than in integron negative isolates, there was no significant correlation between most antibiotics used and integron-positive isolates in our study. Therefore, the use of inappropriate antibiotics, selective pressure, and other factors may be considered for high resistance to antibiotics. The results of our finding describe the importance of class 1 and 2 integrons in multiple antibiotic resistance and its association with MDR P. aeruginosa isolates. Therefore, integrons play an important role in acquisition and dissemination of antibiotics resistance genes among these pathogens, so, management of infection control policies and the appropriate use of antibiotics is necessary for control the spreading of antibiotics resistance genes.

Limitations
The lack of investigation and characterization of resistance gene cassettes associated with class 1 and 2 integrons can be mentioned as one of the main limitations of the present study.