|Year : 2014 | Volume
| Issue : 4 | Page : 229-232
Detection of AdeABC efflux pump genes in tetracycline-resistant Acinetobacter baumannii isolates from burn and ventilator-associated pneumonia patients
Maryam Beheshti1, Malihe Talebi2, Abdollah Ardebili2, Abbas Bahador3, Abdolaziz Rastegar Lari4
1 Department of Microbiology, Iran University of Medical Sciences; Department of Microbiology, Tehran University of Medical Sciences, Tehran, Iran
2 Department of Microbiology, Iran University of Medical Sciences, Tehran, Iran
3 Department of Microbiology, Tehran University of Medical Sciences, Tehran, Iran
4 Department of Microbiology, Iran University of Medical Sciences; Department of Microbiology, Tehran University of Medical Sciences; Razi Drug Research Center, Iran University of Medical Sciences, Tehran, Iran
|Date of Submission||03-Jul-2013|
|Date of Decision||10-Sep-2013|
|Date of Acceptance||16-Feb-2014|
|Date of Web Publication||16-Oct-2014|
Prof. Abdolaziz Rastegar Lari
Department of Microbiology, Iran University of Medical Sciences; Department of Microbiology, Tehran University of Medical Sciences; Razi Drug Research Center, Iran University of Medical Sciences, Tehran
Source of Support: UGC, New Delhi, India,, Conflict of Interest: None
| Abstract|| |
Purpose: Acinetobacter baumannii is the most prevalent nosocomial pathogen which have been emerged in the past three decades worldwide. The aim of this study was to assess the distribution of the AdeABC efflux pump genes, associated with tetracycline resistance in Acinetobacter baumannii isolates collected from burn infection and Ventilator Associated Pneumonia (VAP). Materials and Methods: Ninety-eight A. baumannii isolates were collected from two different hospitals in Tehran, Iran. Tetracycline susceptibility testing was performed by disk diffusion and agar dilution methods according to the CLSI guidelines. The presence of adeSR, adeB, drug efflux system genes in resistant isolates was assessed by polymerase chain reaction (PCR). Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as a chemical inhibitor agent to assess the contribution of AdeABC efflux pump in tetracycline resistance isolates. Results: Approximately 48% (47 out of 98) of isolates showed resistance to tetracycline which 14 (14.2%) isolates were corresponded to burn infection and the remaining 33 (33.8%) strains were isolated from VAP. All tetracycline resistant isolates have AdeABC in PCR assay. The reduction of tetracycline MICs by using 50 μg/ml CCCP were as follows: in 18 isolates 2-4 fold reduction in MICs, 26 isolates showed 8 fold reduction,1 isolate showed 16 fold, 1 isolate showed 32 fold and the remaining 1 isolate showed 128 fold reduction in MICs. Conclusion: The results showed significant correlation between tetracycline resistance and AdeABC efflux pump genes in resistant A. baumannii isolates.
Keywords: Acinetobacter baumannii , burns, efflux pump genes, Iran, tetracycline, ventilator associated penumonia
|How to cite this article:|
Beheshti M, Talebi M, Ardebili A, Bahador A, Lari AR. Detection of AdeABC efflux pump genes in tetracycline-resistant Acinetobacter baumannii isolates from burn and ventilator-associated pneumonia patients. J Pharm Bioall Sci 2014;6:229-32
|How to cite this URL:|
Beheshti M, Talebi M, Ardebili A, Bahador A, Lari AR. Detection of AdeABC efflux pump genes in tetracycline-resistant Acinetobacter baumannii isolates from burn and ventilator-associated pneumonia patients. J Pharm Bioall Sci [serial online] 2014 [cited 2020 Apr 5];6:229-32. Available from: http://www.jpbsonline.org/text.asp?2014/6/4/229/142949
Acinetobacter baumannii is an important opportunistic pathogen causing a variety of nosocomial infections, including bacteremia, urinary tract infection, ventilator-associated pneumonia (VAP), and wound infections. , The high prevalence of wide spectrum antibiotics resistance, because of the presence of intrinsic and acquired mechanisms in A. baumannii may lead to complicated treatments against this organism. ,,,
In spite of the emergence of resistant bacteria, tetracyclines are still the most commonly used antimicrobial agents worldwide.  Tetracycline resistance can be occurred among bacteria because of several mechanisms such as an efflux pump, a ribosomal protection system and enzyme modification. ,
Efflux genes have been reported to be existed in both gram-negative and gram-positive bacteria. Recently, RND family efflux pumps, the AdeABC system, have been shown to be associated with reduced susceptibility and/or resistance to tetracyclines and many other antibiotics in A. baumannii. 
The RND family genes are organized as an operon includes three structural genes which are coding three principle proteins. AdeB is the multidrug transporter which is coded by adeB gene and interact with two other proteins, a membrane fusion protein (MFP) AdeA and outer membrane protein (OMP) adeC which are coded by adeA and adeC genes, respectively. , This interaction allows the antimicrobial agent to pass across the inner and the outer membranes of the bacteria without accumulating in the periplasm. The expression of the AdeABC pump is under the control of the chromosomal adeS (sensor kinase) and adeR (regulator) genes. , Multiple point mutations in adeR and adeS genes have been reported resulting in pump overexpression and subsequently generate multidrug resistance strains (MDR) in A. baumannii. 
In spite of the importance of resistance to tetracycline among clinical isolate, a few data are available on the phenotypic and genotypic characterization of tetracycline among multi drug resistance A. baumannii (MDRAB) infections. Because of limited use of tetracyclines in MDRAB infections, resistance to these antibacterial agents is not routinely monitored.  According to published data there are insignificant documents about the role of the efflux pumps among tetracycline resistant A. baumannii isolates in Iran. The aim of this study was the investigation of AdeABC efflux system genes distribution and their function in tetracycline resistant A. baumannii isolates.
| Materials and Methods|| |
Bacterial isolates and laboratory identification
A total of ninety eight A. baumannii isolates were collected from burn infection in Motahari hospital (65 isolates) and VAP in RasoulAkram hospital (33 isolates) in Tehran during August 2010 to July 2011. Bacterial isolates were identified as A. baumannii using biochemical reactions, including oxidase test, Triple Sugar Iron (TSI), SIM and Oxidation-Fermentation (OF) test  and finally confirmed by PCR amplification of blaOXA-51-like gene. 
Antimicrobial susceptibility tests
The susceptibility of A. baumannii isolates to tetracycline (30 μg) (Mast, Merseyside, U.K) was tested using the standard agar disk diffusion and minimum inhibitory concentration (MIC) of tetracycline was performed by agar dilution method on Mueller-Hinton (M-H) agar (Merck, Germany) according to the Clinical and Laboratory Standard Institute (CLSI).  Tetracycline was purchased from Sigma, Aldrich Germany. A. baumannii ATCC 19606 and Pseudomonas aeruginosa ATCC 27853 strains was used as quality control references for susceptibility testing.
PCR analysis of AdeABC efflux pump and nucleotide sequencing
The standard PCR assay was performed using specific primers for detection of structural adeB gene and two regulatory genes, adeR and adeS belong to AdeABC system in tetracycline-resistant A. baumannii isolates the DNA amplification instrument Mastercycler gradient to detect one structural (adeB) and two regulatory (adeR and adeS) genes of AdeABC. The genomic DNAs for PCR were prepared by boiling method. Three pairs of oligonucleotide primers used for the reactions are listed in [Table 1]. The PCR was performed in a reaction mixture with total volume of 25 μl containing 2x Master Mix Red (Ampliqon, Denmark) (containing 10 pmolTaq DNA Polymerase), 0.5 mM of each primer and 1 μl of DNA suspension.The PCR program was performed as follows: Initial denaturation at 94°C for 5 minutes; 30 cycles containing of denaturation at 94°C for 1 minute, annealing at 57°C for 1 minute, and extension at 72°C for 1 minute; followed by a final extension at 72°C for 5 minute. The amplified products were subjected to electrophoresis in 1% agarose gels.
Inhibitory effects of CCCP
Efflux pump system function was evaluated by MICs to tetracycline with and without treatment of efflux pumps inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Sigma-Aldrich Germany) according to modified method performed in previous studies. , Briefly, 50 μg/ml of CCCP was added into each M-H agar plate containing tetracycline concentration from 0.5 to 1024 μg/ml. Then, MIC of tetracycline was determined for all clinical isolates. A. baumannii ATCC 19606 was used as a control and plates without CCCP were used as negative control. The inhibitory effect of CCCP on efflux pump should be determined by two-fold or greater reduction in MICs value after treatment with CCCP. 
| Results and Discussion|| |
Bacterial isolates and antimicrobial susceptibility assay
Antibiotic resistance is a major challenge in treating infections caused by gram negative bacteria, including A. baumannii .  According to various studies, the rate of mortality because of MDR A. baumannii infections in patients hospitalized in burn and intensive care units has been increased.  In fact, the huge spread of these bacteria that are responsible for nosocomial infections is of great concern worldwide. 
As shown in [Table 1] and [Table 2], approximately 48% (47out of 98) of isolates showed resistance to tetracycline which 14 (14.2%) isolates were corresponded to burn infection and the remaining 33 (33.8%) strains were isolated from VAP [Table 2] and [Table 3]. In the present study, the rate of resistance among burn samples was lower than ventilator associated pneumonia (VAP). It indicates that resistant strains significantly belong to VAP samples. Resistance rate observed in this study was much lower than other reports from USA, UK and Italy with 85.5%, 84% and 75%, respectively, ,, but was more than the rate (34.7%) reported by Asadollahi and colleagues.  Nevertheless, our susceptibility test results are similar to the results (55%) reported by Yagang Chen and colleagues.  Although the widespread prevalence of resistant Acinetobacter spp. in hospital particularly in Intensive Care Units (ICUs) is due to high resistance rate of these organisms to environmental conditions but there are predisposing factors in acquisition of resistant strains infections significantly including mechanical ventilation and surgical procedures. 
|Table 2: Distribution of the MIC for tetracycline in burnclinical isolates of A. baumannii before and after treatment with CCCP|
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|Table 3: Distribution of the MIC for tetracycline in VAP clinical isolates of A. baumannii before and after treatment with CCCP|
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PCR amplification of the AdeABC efflux system
The AdeABC efflux pump is one of the principle antibiotic resistance mechanisms which are applied by Gram-negative bacteria such as A. baumannii . Prevalence of MDR Acinetobacter spp. is mediated by overexpression of efflux pump and antibiotic resistant genes. ,,,,, Genetic detection of RND genes can be used for identification of resistant Acinetobacter spp. and A. baumannii strains with lack of adeABC genes possibly are susceptible to antimicrobial agents.  In vitro studies showed that tetracycline has efficient activity against A. baumannii nevertheless is not prescribed in clinical use or for VAP treatment commonly.  In recent studies, it has been proved that 80% of MDR A. baumannii isolates showed close correlation between resistance and AdeABC pump genes.  It was proved that AdeABC pump is responsible for increasing resistance to common antibiotics which are as substrates for efflux pump such as tetracycline. 
PCR assay was performed with specific primers as described above. The expected amplification fragment with sizes of approximately 541 bp, 447 bp and 544 bp were detected on agaros gel corresponded to adeB, adeR and adeS genes, respectively in all tetracycline resistant isolates [Figure 1]. In the present study harboring the tetracycline resistance genes (AdeB, AdeR, AdeS) was confirmed in all resistant strains.
|Figure 1: PCR amplification of adeB, adeS and adeR genes in five selected isolates of A. baumannii. (a) Lane1: Positive control for adeB; lane 2-5: PCR product of adeB gene (541 bp); M: 1kb DNA size marker. (b) Lane1: Positive control for adeR; lane2-5: PCR product of adeR gene (447 bp); M: 1kb DNA size marker. (c) Lane1: positive control for adeS Lane 2-5: PCR product of adeS gene (544 bp); M: 1kb DNA size marker|
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Inhibitory effects of CCCP on MICs of tetracycline
Moreover, we found that MICs of tetracycline decreased mainly 2 to 8 fold in the most resistant isolates (44 out of 47) in the presence of CCCP. These results suggested that active efflux pumps contribute in resistance to tetracycline in A. baumannii isolates.
The MIC range of tetracycline in all resistant A. baumannii isolates were between 32 to 1024 μg/ml. In burns samples, the MIC of 6 (43%) and 8 (57%) of isolates were 128 and 256 μg/ml respectively. The reduction of tetracycline MICs by using 50 μg/ml CCCP were as follows: In 8 (57%) isolates 2-4 fold reduction in MICs, 4 (29%) isolates showed 8 fold reduction, 1 (7%) isolate showed 32 fold and the remaining 1 (7%) isolate showed 128 fold reduction in MICs [Table 2].
MICs values of tetracycline in VAP isolates were 32 μg/ml in 2 out of 33 (6%) isolates and for the remaining 31 (94%) isolates were 256≤μg/ml. Tetracycline MICs with using CCCP were measured in VAP and the results indicated 2-4 fold reduction in 10 isolates, 8 fold reduction in 22 isolates and 16 fold reduction in the remaining 1 isolate [Table 3]. It is worth mentioning that burn isolates showed 2-128 fold reduction while VAP isolates showed 2-16 fold reduction in tetracycline resistance.
Our comprehensive analysis revealed that criteria of infection diagnosis and antibiotic consumption play an important role in prevention and control of A. baumannii infections. There are significant factors needs to be measured in order to control the appearance of MDR A. baumannii strains including survey resistance mechanisms, use of new drugs, and avoidance of the widespread of multiresistant isolates. Using standard assays for susceptibility test and MIC breakpoint could be carried out to accurately monitor of resistant strains.  In addition, Acinetobacter baumannii is one of the most principle agents in nosocomial and burn infection thus exact diagnosis of isolates, antibiotic resistant pattern and the mechanisms of resistant can be so helpful for prevention or controlling the infections.
| Acknowledgment|| |
This study was supported by Tehran University of Medical Sciences, grant no. 91-01-30-16016.
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[Table 1], [Table 2], [Table 3]
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