Acinetobacter is a gram-negative, non-motile, obligate aerobic, oxidase-negative, and non-fermenting rod belonging to the Moraxellaceae family. This bacterium is an important opportunist, responsible for nosocomial infections, especially in intensive care units (ICUs), which can produce various types of infection such as ventilator-associated pneumonia, bacteremia, surgical-site infections, secondary meningitis, and urinary tract infections.1,2 Acinetobacter spp. are predominantly found in soil, water, food, animals, and humans, while its existence on surfaces of healthcare environments has been difficult to control.3 The recent emergence of multidrug-resistant (MDR) Acinetobacter baumannii has become a serious problem in clinical settings and these MDR strains are spreading rapidly among hospitalized patients.4 Several exchangeable genetic elements such as plasmids, transposons, and integrons are the most important genetic elements responsible for the transmission of antibiotic resistance genes in different gram-negative bacteria.5 Various studies showed that integrons are significantly in relation to the presence of MDR in A. baumannii isolates.3,4 These elements play a major role in the dissemination and rearrangement of resistance determinants called mobile genetic cassettes.6 So far, several classes of integrons have been described in gram-negative bacteria.7 All integrons have a 5’conserved segment, consist of the integrase gene, and the cassette integration site (attI), but have a distinct 3’conserved segment.8 The most prevalent integrons belong to class Iand play an important role in the development of antimicrobial resistance and the emergence of MDR profiles in gram-negative bacteria. As for the class I integrons, the 3’conserved sequence area (3’CS) includes three open reading frames: qaEΔ1 gene which confers resistance to antiseptic compounds, a sul1 gene which confers resistance to sulfonamides and ORF5, of unknown function.3,9 Integrons of class II are commonly found associated with the Tn7 transposon family and its 3’conserved segment containing five tns genes, which are responsible for the mobility of transposons. Class II integrons have been described most often in isolates within the Enterobacteriaceae family. Integrons of class III have also been reported but its 3’conserved segment has not been characterized.9,10 The prevalence of different classes of integrons and their relationship with antibiotic resistance in clinical isolates of A. baumannii is not clear in Iran and different parts of the world. Due to the emergence of a high prevalence of multiple and extensive drug resistance (XDR) A. baumannii isolates in our region, the objectives of this study were to investigate the prevalence of class I–III integrons among A. baumannii isolates collected from hospitalized patients.
Methods
We performed a cross-sectional study from October 2015 to October 2016 at two teaching hospitals affiliated to Isfahan University of Medical Sciences, Isfahan, Iran. The non-duplicated A. baumannii isolates were obtained from various clinical specimens such as blood, wounds, and urine, and were transported to the microbiology 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 A. baumannii using standard microbiological methods, including Gram staining, oxidative or fermentative metabolism, catalase, oxidase, motility, and production of acid from different sugars and genotypic method (the presence of the blaOXA-51 gene).11 The confirmed isolates were stored at -80 °C in brain heart infusion broth containing 20% glycerol.
The antibiotic susceptibility pattern was determined based on disk diffusion method on Mueller–Hinton agar (Himedia, India) according to the Clinical and Laboratory Standards Institute (CLSI) recommendation for imipenem (10 µg), meropenem (10 µg), ertapenem (10 µg), cefepime (30 µg), ceftazidime (30 µg), ceftriaxone (30 µg), piperacillin-tazobactam (100/10 µg), gentamicin (10 µg), amikacin (30 µg), ciprofloxacin (5 µg), co-trimoxazole (25 μg), and tetracycline (30 µg) disks (MAST, Merseyside, UK).
The CLSI interpretive criteria for Pseudomonas aeruginosa was applied to determine the susceptibility to polymyxin B and colistin. P. aeruginosa ATCC 27853 were used as standard quality controls.12 MDR and XDR were estimated according to previously described definitions.13
A phenol-chloroform method was used to extract genomic DNA as described previously.14 The extracted DNA were dissolved in 100 μl sterile distilled water and stored at -20 oC. The polymerase chain reaction (PCR) was performed to detect blaOXA-51 and the presence of class I, II, and III integrase genes using the specific primers.15
The conditions for PCR amplification were: initial denaturation at 94 °C for 5 minutes, followed by 35 cycles of denaturation at 94 °C for 45 seconds, primer annealing at 54 °C for intI1 and intI2 and 52 °C for intI3 and extension at 72 °C for 45 seconds, and a final extension at 72 °C for 7 minutes.
Amplification products were analyzed using 1.5% agarose gel with KBC power load dye (CinnaGen Co. Iran). Positive results were confirmed by direct sequencing of the PCR products.
Statistical analysis was performed using SPSS Statistics (IBM Corp. Released 2012. IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp.). The results are presented using descriptive statistics in terms of relative frequency. Fisher’s exact test was used to determine any statistical association. Statistical significance was regarded as p-values < 0.050.
Results
A total of 147 confirmed A. baumannii isolates were collected from different clinical samples of studied hospitals. Overall, 95 (62.5%) A. baumannii isolates were obtained from male and 57 (37.5%) from female samples. Table 1 shows the distribution of integron-positive isolates based on clinical specimens and different wards. Accordingly, most of the integron-positive isolates were obtained from the trachea followed by sputum. Of the 147 isolates, 97.3% (143/147) of A. baumannii isolates were identified as XDR and 2.7% (4/147) of isolates were MDR.
PCR amplification of the three classes of integron genes revealed that 94 isolates (63.9%) carried class I integrons, 115 isolates (78.2%) harbored class II integrons, and 73 isolates (49.6%) had both classes of integron genes. Class III integron was not found in any isolates [Figure 1]. The results also showed that all integron-positive isolates were resistant to imipenem, meropenem, and ceftriaxone and the most effective antibiotics against integron-positive isolates were colistin.
Table 1: Distribution of integron-positive Acinetobacter baumannii isolates based on clinical specimens and different wards.
|
Wards |
|
|
|
ICU |
58 (61.7) |
79 (68.7) |
|
Emergency |
12 (12.8) |
9 (7.8) |
|
Surgery |
13 (13.8) |
14 (12.2) |
|
Internal medicine |
11 (11.7) |
13 (11.3) |
|
Clinical sample |
|
|
|
Trachea |
48 (51.1) |
64 (55.7) |
|
Urine |
4 (4.3) |
5 (4.3) |
|
CSF |
10 (10.6) |
9 (7.8) |
|
Wound |
11 (11.7) |
13 (11.3) |
|
Sputum |
13 (13.8) |
14 (12.2) |
|
Blood |
3 (3.2) |
4 (3.5) |
ICU: intensive care unit; CSF: cerebrospinal fluid.
The antimicrobial resistance patterns of integron-positive and integron-negative isolates are shown in Tables 2 and 3. We observed a significant correlation between the presence of class I and class II integron with higher rate of resistance related to some antibiotics such as imipenem, meropenem, and ceftazidime (p-value < 0.001).
Table 2: Antibiotic susceptibility pattern of class I integron-positive and integron-negative of Acinetobacter baumannii strains.
|
Carbapenems |
Imipenem |
94 (100) |
- |
52 (98.1) |
1 (1.8) |
< 0.001 |
|
Meropenem |
94 (100) |
- |
51 (96.2) |
2 (3.7) |
< 0.001 |
|
Ertapenem |
94 (100) |
- |
53 (100) |
- |
1.000 |
|
Cephalosporins |
Cefepime |
93 (98.9) |
1 (1.0) |
53 (100) |
- |
1.000 |
|
Ceftriaxone |
94 (100) |
- |
53 (100) |
- |
1.000 |
|
Ceftazidime |
90 (95.7) |
4 (4.2) |
52 (98.1) |
1 (1.8) |
< 0.001 |
|
β-lactam/β-lactamase inhibitor |
Piperacillin/tazobactam |
92 (97.8) |
2 (2.1) |
53 (100) |
- |
< 0.001 |
|
Tetracyclines |
Tetracycline |
66 (70.2) |
28 (29.7) |
50 (94.3) |
3 (5.7) |
0.001 |
|
Fluoroquinolones |
Ciprofloxacin |
94 (100) |
- |
52 (98.1) |
1 (1.8) |
< 0.001 |
|
Aminoglycosides |
Amikacin |
85 (90.4) |
9 (9.5) |
47 (88.6) |
6 (11.3) |
< 0.001 |
|
Gentamycin |
89 (94.6) |
5 (5.3) |
50 (94.3) |
3 (5.7) |
1.000 |
|
Sulfonamide |
Co-trimoxazole |
92 (97.8) |
2 (2.1) |
52 (98.1) |
1 (1.8) |
1.000 |
Table 3: Antibiotic susceptibility pattern of class II integron-positive and integron-negative of Acinetobacter baumannii strains.
|
Carbapenems |
Imipenem |
115 (100) |
- |
31 (96.8) |
1 (3.1) |
< 0.001 |
|
Meropenem |
115 (100) |
- |
31 (96.8) |
1 (3.1) |
< 0.001 |
|
Ertapenem |
114 (99.1) |
1 (0.8) |
32 (100) |
- |
1.000 |
|
Cephalosporins |
Cefepime |
115 (100) |
- |
31 (96.8) |
1 (3.1) |
< 0.001 |
|
Ceftriaxone |
115 (100) |
- |
32 (100) |
- |
< 0.001 |
|
Ceftazidime |
112 (97.3) |
3 (2.6) |
30 (93.7) |
2 (6.2) |
< 0.001 |
|
β-lactam/β-lactamase inhibitor |
Piperacillin/tazobactam |
114 (99.1) |
1 (0.8) |
32 (100) |
- |
1.000 |
|
Tetracyclines |
Tetracycline |
109 (94.7) |
6 (5.2) |
28 (87.5) |
4 (12.5) |
< 0.001 |
|
Fluoroquinolones |
Ciprofloxacin |
114 (99.1) |
1 (0.8) |
32 (100) |
- |
1.000 |
|
Aminoglycosides |
Amikacin |
101 (87.8) |
14 (12.1) |
31 (96.8) |
1 (3.1) |
< 0.001 |
|
Gentamycin |
107 (93.0) |
8 (6.9) |
31 (96.8) |
1 (3.1) |
< 0.001 |
|
Sulfonamide |
Co-trimoxazole |
113 (98.2) |
2 (1.7) |
31 (96.8) |
1 (3.1) |
< 0.001 |
Discussion
The spread of clinical multidrug resistance A. baumannii isolates with high resistance to different classes of antibiotics has become a serious problem.16 Resistance to antimicrobial agents is often associated with the horizontal transmission of antimicrobial resistance genes via mobile elements, such as plasmids and transposons.17 Therefore, the emergence of the antimicrobial resistance genes through integrons in MDR A. baumannii isolates has become a major concern in the treatment of infections caused by these bacteria.
In recent years, this is the first study addressing the emergence of XDR A. baumannii isolates based on a standard definition for these isolates in our region, and there are few studies on the frequency of XDR and MDR isolates. Our finding revealed a high rate of XDR A. baumannii isolates (97.3%; 143/147), which were resistant to several antimicrobial agents used for the treatment of A. baumannii infections.
The rate of XDR isolates in our region is close to the study conducted by Jasemi et al,18 in which the rate of XDR isolates was detected in 91.3%
from Iranian healthcare settings. However, the frequency of XDR isolates described by Jasemi et al (60%),18 Maspi et al (71.2%),19 and Fazeli et al (62.8%)20 are slightly lower than our finding. However, the high incidence of XDR isolates may be related to misdiagnosis and subsequent useless antibiotic prescriptions in our healthcare setting.
Due to high rate of antibiotic resistance in our isolates, there were no correlation between distribution of antibiotic resistance and different clinical samples. However, the most antibiotic resistance were among respiratory samples where A. baumannii are a major cause of respiratory infections.
Our data indicated 63.9% and 78.2% of A. baumannii isolates carried intI1 and intI2 integron-integrase genes, respectively. Meanwhile, the intI3 integron-integrase gene was not detected in any isolates.
Numerous studies have been investigating the presence of class I and II integrons in clinical A. baumannii isolates from Iran and other parts of the world that it is important to note that in most of these studies, the higher prevalence of class I integron was reported, however, our study indicates otherwise.21–24
In contrast to previous data, our finding may explain the emergence of the high prevalence of intI2 for the first time in our region, which indicates a concern in the future and increase the antibiotic resistance associated with the presence of class II integron. Closest to our finding, in a study conducted by Mirnejad et al,3 the prevalence of class I and class II integrons was reported in 42% (21/50) and 82% (41/50) of isolates. Ramírez et al,16 reported the high prevalence of the intI2 gene (68%) in clinical samples isolated from Buenos Aires City, Argentina. Kamalbeik et al,25 also reported 65% of the clinical isolates A. baumannii were classified as class II integrons. The previous study indicates the rate of incidence of class I in A. baumannii isolates is variable due to geographical distribution and origin of infections. Goudarzi et al,26 showed that 74.1% and 12.5% of A. baumannii isolates harboring class I and II integrons, respectively. Another Iranian study showed 96.7% and 43.3% of XDR A. baumannii isolates were positive for class I and class II integrons, respectively.27
According to antibiotic susceptibility pattern, the presence of class I and class II integron showed a significant correlation with higher rates of antibiotic resistance with most antibiotics used for in vitro compared to integron-negative isolates.
In addition, we compared the antimicrobial resistance pattern. The most effective antibiotic against integron-negative and integron-positive isolates were polymixins with 100% susceptibility and followed by tetracyclines for class I integron-positive and amikacin for class II integron-positive isolates.
Our results are in agreement with previous reports conducted by Mirnejad et al,3 Japoni-Nejad et al,28 and Chen et al,29 which have shown the significant differences with an increased risk of antibiotic resistance in relation to the presence of a different class of integrons.30
These associations demonstrating the important role of integrons in antibiotic resistance and thereby in the epidemic behavior of A. baumannii. It should be noted that the resistance of integron-negative A. baumannii isolates is probably due to other mechanisms of antibiotics resistance.
Conclusion
Our findings highlight the emergence of class II integrons. Moreover, results of our study contribute to the understanding of the distribution of class I and II integrons in A. baumannii and its association with MDR and XDR isolates in our region. The use of appropriate infection control measured in clinical settings and implementation of treatment strategies is very necessary for our health care setting.
Disclosure
The authors declared no conflict of interest. No funding was received for this study.
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