Background and Objective: The acquisition and dissemination of antibiotic-resistant agents in Acinetobacter baumannii (A. baumannii) is highly facilitated through integrons that are DNA transposable elements and able to receive genes through site-specific recombination. Class 1 and 2 integrons are the most known integrons that are frequently available in A. baumannii. The aim of this study was to determine the frequency of class 2 integron in multidrug resistance (MDR) A. baumannii. Methods: A total of 100 isolates of A. baumannii were collected from patients that are admitted to hospitals in Kermanshah in the year 2014-2015. The isolates were identified using biochemical tests and the kit API 20 NE, and then their sensitivity to 20 antibiotics was examined. The prevalence rate of class 2 integrons among the isolates was determined using Polymerase chain reaction (PCR). The results were analyzed using Fisher’s test and the non-parametric Chi-squared test. Results: The maximum drug resistance was observed against Cefotaxime (93%), Ceftriaxone (92%), Mezlocillin (91%), Ceftazidime (86%), Imipenem (82%), and Piperacillin (82%). The minimum drug resistance was observed against Colistin (12%), Polymyxin B (7%), Minocycline (10%), and Tigecycline (6%) and seems as the most efficient antibiotics. Twenty-nine (29%) isolates out of the 100 isolates were multidrug resistance (MDR); about 21 isolates (21%) were extensive drug resistance (XDR); and none were pan drug resistance (PDR). Thirty four (34%) isolates contained class 2 integrons. The results did not showed a significant correlation between the presence of class 2 integrons and incidence of MDR A. baumannii. Conclusion: The transmission of antibiotic-resistant agents by the class 2 integrons resulted in further durability and spread of these isolates in the environment. Therefore, it seems that resistance to A. baumannii is always changing, and different countries should attend to these changes.
Keywords: Acinetobacter baumannii, class 2 Integron, extensively drug-resistant, multi-drug resistant, pandrug-resistant
Acinetobacter baumannii (A. baumannii) is one of the most important nosocomial pathogens which particularly affect critically ill patients in intensive care units (ICU)., This pathogen is known for causing variety of nosocomial infections, including bacteremia ventilator-associated pneumonia, surgical-site infections, urinary tract infections, and secondary meningitis., A. baumannii is becoming a serious clinical concern because of its noticeable ability to develop resistance to several classes of antimicrobial drugs. There are also increasing reports of multidrug resistant of A. baumannii (MDRAB) outbreaks in different clinical settings inworldwide., The MDRAB term was defined as those isolates that are intermediate or resistant to at least three different classes of antimicrobial agents mainly beta-lactamas, aminoglycosides, fluoroquinolones, andcarbapenems. In recent years a novel mechanism for dissemination of resistance gene among bacteria has beendescribed. This mechanism is based on mobile elements, including plasmids transposons and integrons. In these antibiotic-resistance mechanisms, integrons are considered as unique for their capacity to cluster and express resistance genes. Resistant gene contributes to the ability of A. baumannii to be such a successful pathogen. Integrons, a mobile DNA elements, are capable of capturing the various genes by a site-specific recombination mechanism that often carry gene cassettes containing antibiotic resistance genes. They are considered unique due to their capacity to cluster and express antimicrobial resistance factors. Several classes of different integrons have been described with class 1 integrons as being the most common and widely distributed among gram-negative bacteria. Second class of integrons are found in transposon, recently, the incidence of integron class 2 has increased. Class 2 integrons are embedded in the Tn7 family of transposons. The Tn7 includes adefective integron consisting of the gene cassettes dfrA1-sat2-aada1-or fX in its variable region., The aim of this study was to determine the frequency of class 2 integron in multidrug resistance (MDR) A. baumannii.
One hundred strains of A. baumannii were identified from patients hospitalized between September 2014 and October 2015 in four hospitals in the Kermanshah region, Iran. These strains were recovered from sputum, abdominal abscesses, blood, urine, wounds and burns, and Synovial fluid. The isolates were stored at-70°C in trypticase soy broth supplemented with 30% glycerol and further pending analyses of all the supplemented colonies were assessed by using standard biochemical tests such as oxidase, TSI (Triple Sugar Iron Agar), OF test (Oxidative Fermentative test) and then after using API 20 NE (version 6.0, bioMerieux, Marcy L Etoile, France) and Growth at 44°C, it has been identified as A. baumannii. The strains isolated were stored at-70°C in nutrient broth containing 30% glycerol v/v for further investigation.
Antimicrobial susceptibility testing
Antimicrobial susceptibility testing was performed on all isolates by the Kirby–Bauer disk diffusion method for the following antimicrobial agents: Amikacin (30 µg), Cefepime (30 µg), Cefotaxime (30 µg), Ceftazidime (30 µg), Ceftriaxone (30 µg), Ciprofloxacin (5 µg), Cotrimoxazole (1/25–23/75 µg), Gatifloxacin (5 µg), Gentamicin (10 µg), Colistin (10 µg), Imipenem (10µg), Levofloxacin (5 µg), Meropenem (10 µg), Mezlocillin (75 µg), Minocycline (30 µg), Piperacillin (100 µg), Polymyxin B (300 unit), Tetracycline (30 µg), Tigecycline (15 µg), Tobramycin (10 µg) using the table of CLSI 2012; and using disk: (MAST, merseyside, UK). A. baumannii ATCC 19606 was used as a reference strain. The MDR was defined resistant to three classes of antibiotics including Cephalosporins (Cefepime and Ceftazidime), Carbapenems (Imipenem and Meropenem), and Quinolones (Ciprofloxacin) and also resistant to carbapenem, called XDR.
Genomic DNA used for PCR assays was obtained from bacterial suspension grown overnight in Luria broth (LB) with shaking incubator at 37°C. Extraction of genomic DNA from A. baumannii isolates was performed by boiling method. Bacterial suspension was centrifuged at 7000 r/min for 10 min. Amplification reaction was performed in a final volume of 25 µL. Each reaction mixture contained 10 × PCR buffer, Taq DNA polymerase, and mixed deoxy ribonucleosid triphosphaes, MgCl2 and primers int 2/F/R were used to amplify 280 bp fragments and suspension bacterial, az described by mirnejad in 2013 for detection of class 2 integron was used for primers 5-Cs and3-Cs. Sequences of each primers for polymerase chain reaction method were mentioned in [Table 1]. The conditions of PCR were 95°C for 5 min, followed by 30 cycle of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 30 sec, and final extension at 72°C for 5 min. The PCR products were separated by electrophoresis on a 1% agarose gel. The electrophoresis conditions were 80 V, 40 min, and finally the gel was stained with ethidium bromide.
In cases when comparison and the relationship between antibiotic resistant pattern and integron positive genotype from statistical tests for measuring P-value is needed, then Fisher’s test and the non-parametric Chi-squared test is used for this point.
A total of about 100 A. baumannii isolates were highly resistant to Ceftriaxone, Cefotaxime, Mezlocillin (>90%), Imipenem, Ceftazidime, Piperacillin (>80%) [Table 2], and susceptible to Colistin, Polymyxine B, Minocycline, Tetracycline. MDR A. baumannii isolates were mainly isolated from sputum (60%), abdominal abscesses (19%), blood (6%), urine (5%), wound (2%), synovial fluid (2%), from male (61%), age (mean = 40.3 ± 25.1, min = 1, max = 83) and female (39%) age (mean: 40.3 ± 25.7, min = 1 , max = 83) and the samples were collected from four hospital in Kermanshah. MDR and XDR frequencies were, respectively, 29% and 21%. The results were analyzed by Chi-sqaured fisher (KI2) using SPSS software (Version.20 IBM, Chicago, IL, USA) and P value (0.05). Based on PCR assays [Figure 1] and [Figure 2], 34 (34%) isolates contained class 2 integrons. Accordingly, there was not related to the incidence of MDR (P value 0.9) with class 2 integron. 5/-cs-3/-cs was the area with variable region and there were the genes cassette integrates and genetic elements such as integrons in this area were, respectively (42, 76%). Levofloxacin, Cefepime, Mezlocillin, Ciprofloxacine, Piperacillin, Cefetaxime, Ceftazidime, Ceftriaxone, Gentamicin a significant increase of resistant in ICU ward [Table 3].
A. baumannii isolates are typically resistant to various antibacterial agents such as Cephalosporines, Aminoglycosides, Penicillins, Macrolides, Fluoroquinolones, and Tetracyclines. The including of MDR related to other genetic factors also. It seems resistance was through the different ways such as deficiency in the enzyme, in the cell wall, or vial plasmid, and chromosome that did not included in our study. The prevalence of MDR and XDR causes epidemic in different geographic area., In our study, frequency of integron 1 was lower than research of Iran such as Taherikalani in 2011 and outside of Irandone. MDR strains from different countries such as Europe, North America, Argentina, Brazil, China, Taiwan, Hong Kong, Japan, Korea, and Iran have been reported. The prevalence of MDR strains can cause epidemics (Outbreak) in cities, countries, and continents. According to the Agency for Health Care in America within a year from 2002 to 2003, the percentage of MDR strains had increased from 7% to 22%. The frequency of MDR isolates was reported 93% among A. baumannii isolates in the previous study of Mohajeri et al., the same as this study. Frequency of MDR isolated was the most frequent isolates in Peymani et al.‘s study. Class 2 integrons are the most common among the clinical Acinetobacterisolates., In our study, 34% of isolates contained class 2. In contrast with Mirnejad’s study, 82% of MDR isolates contained class 2. In a study in the year 2003–2009 on 97 A. baumannii at United States Army Medical Building, 80% of isolates were MDR, the same as the current study.
In a study by Jafari et al. in 2012 in Fars hospitals frequency for MDR 97% and XDR was equal to 56% and zero PDR, but in this study the prevalence of strains of MDR 29% and prevalence of strains XDR 21%, and none were PDR.
The transmission of antibiotic-resistant agents by class 2 integrons resulted in further durability and spread of these isolates in the environment. Therefore, it seems that resistance to A. baumannii is always changing, and different countries should attend to these changes. Infections with MDR isolates in ICU are serious problems that can compromise patient’s survival and the outcome of reconstructive treatment. Nevertheless, further studies need to be carried out to characterize other genetic elements with integrons in these isolates with MDR and XDR phenotype and association of integrons with epidemic patterns that will be obtained by molecular typing methods such as multilocus sequence typing (MLST) and pulsed field gel electrophoresis.
We gratefully acknowledge the Vice-Chancellor for Research and Technology, Kermanshah University of Medical Sciences, for financial support of this study resulting from MSc Microbiology thesis of Rozhin Souzani, Kermanshah University of Medical Sciences, Iran (Grant No.95213).
Financial support and sponsorship
Kermanshah University of Medical Sciences.
Conflicts of interest
There are no conflicts of interest
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]