|Year : 2020 | Volume
| Issue : 1 | Page : 205-209
Role of AdeB gene in multidrug-resistance Acinetobacter
Rawheia H Aladel1, Soheir A Abdalsameea2, Hanem M Badwy1, Shimaa A Refat3, Reem M ElKholy1
1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Clinical Biochemistry and Diagnostic Molecular Biology, National Liver Institute, Menoufia University, Menoufia, Egypt
3 Department of Clinical Pathology, Faculty of Medicine, Banha University Banha, Egypt
|Date of Submission||31-Dec-2018|
|Date of Decision||17-Jan-2019|
|Date of Acceptance||10-Feb-2019|
|Date of Web Publication||25-Mar-2020|
Reem M ElKholy
ShebinElkom, Faculty of Medicine, Yaseen Abdelghafar Street 32511
Source of Support: None, Conflict of Interest: None
The aim was to determine the role of the Ade B gene in multidrug-resistant Acinetobacter isolated from ICUs of Menoufia University Hospitals.
Acinetobacter is a gram-negative bacteria that may cause serious infections. Numerous mechanisms are involved in its resistance to drug therapy. The active efflux mechanism is an important factor for development of multidrug resistance. The Ade ABC system is important efflux system in mediating such resistance. Therefore, the present study was designed to analyze the association between the expression level of Ade B gene and drug resistance in Acinetobacter.
Materials and methods
This case–control study was carried out in the period between October 2016 and October 2018. It was done at Clinical Pathology Department, Faculty of Medicine, Menoufia University Hospitals. The patients were selected from ICUs of Menoufia University Hospitals. The study included clinical samples collected from 614 patients admitted to ICUs. All clinical Acinetobacter isolates were further studied for determination of antibiotic susceptibility patterns and detection of Ade B gene by real-time PCR.
Of the 614 samples, 70 (11.4%) Acinetobacter were isolated. Regarding antimicrobial resistance pattern, 61.4% of the Acinetobacter isolates were found multidrug and extensive drug resistant. There was significant increase in Ade B gene expression (P < 0.001) in multidrug-resistant isolates in relation to susceptible isolates.
AdeB gene plays a vital role in multidrug resistance in clinical Acinetobacter isolates. These results may benefit to design active efflux pump inhibitors. Moreover, implementation of strict microbial policies and infection control programs may prevent the rapid dissemination of this organism.
Keywords: Acinetobacter, Ade ABC system, efflux Pumps, multidrug resistance, nodulation-division
|How to cite this article:|
Aladel RH, Abdalsameea SA, Badwy HM, Refat SA, ElKholy RM. Role of AdeB gene in multidrug-resistance Acinetobacter. Menoufia Med J 2020;33:205-9
|How to cite this URL:|
Aladel RH, Abdalsameea SA, Badwy HM, Refat SA, ElKholy RM. Role of AdeB gene in multidrug-resistance Acinetobacter. Menoufia Med J [serial online] 2020 [cited 2020 Mar 30];33:205-9. Available from: http://www.mmj.eg.net/text.asp?2020/33/1/205/281307
| Introduction|| |
Acinetobacter is an oxidase-negative and catalase-positive nonmotile gram-negative pathogen. The emergence of antibiotic resistance among Acinetobacter in hospitalized patients is a serious and recurrent problem for the treatment of infections. It is responsible for infections in skin and soft tissue, bloodstream, meningitis, urinary tract infection, pneumonia, ventilator-associated pneumonia, and endocarditis. Patients in intensive care units and immunocompromised patients are at high risk for acquiring this pathogen.
The resistant mechanisms of Acinetobacter include formation of inactivated enzymes, gene mutations in chromosomes, changes in outer membrane porins, and the active drug efflux mechanism.
The efflux system is a mechanism of antibiotic resistance involving the extrusion of toxic substrates from within cells into the external environment with the help of transport proteins. In the bacteria kingdom, multidrug transporters can be divided into five major families.
The RND-type superfamily is the most commonly found efflux system in gram-negative bacteria including Acinetobacter species. The Ade ABC efflux pump belongs to the RND-type superfamily, which consists of Ade A (membrane fusion), Ade B (multidrug transporter), and Ade C (outer membrane) genes. The main role of Ade B gene resulted from its function; Ade B captures its substrates either from the cytoplasm or within the phospholipid bilayer of the inner membrane in Acinetobacter, whereas Ade A and Ade C act as assistance. So this study was designed to determine the role of the Ade B gene in multidrug-resistant Acinetobacter isolated from ICUs of Menoufia University Hospitals.
| Materials and Methods|| |
This case–control study was carried out in the period between October 2016 and October 2018. It was done at Clinical Pathology Department, Faculty of Medicine, Menoufia University Hospitals. The patients were selected from ICU of Menoufia University Hospitals. Different samples including bronchial aspirate, endotracheal aspirate, sputum, blood, urine, burn swab, and pus were collected from 614 patients who were referred to ICU of Menoufia University Hospitals. Each sample was subjected to culture and sensitivity to detect Acinetobacter.All the selected patients were subjected to personal history (name, age, and sex), clinical history (date, cause of hospital admission, central venous lines, or endotracheal tubes), associated comorbidities (diabetes, hypertension, obesity, chronic lung diseases, chronic renal diseases, chronic liver diseases, immunosuppression, under corticosteroids therapy, or chemotherapy), administration of antimicrobial agents, and length of hospital stay before sampling. Colony isolates were obtained from culture of samples on blood agar and MacConkey agar (Oxoid Cop., Cheshire, England). Each nonlactose fermenting colony on MacConkey agar media was picked up. Acinetobacter isolates were identified by microscopic examination using Gram stain, culture characteristics, conventional biochemical reactions (catalase, oxidase, indole, urease, and ornithine decarboxylase), and Vitek −2 compact system. Susceptibility screening tests were done for all Acinetobacter isolates by disk diffusion method against different antimicrobial agents. Mueller Hinton agar plates were inoculated by 0.5 McFarland turbidity suspensions for each Acinetobacter isolate. Plates were incubated at 35 ± 2°C in ambient air for 20–24 h. according to clinical and laboratory standard institute guidelines (CLSI, 2018). Results were categorized as susceptible, intermediate, and resistant. Acinetobacter baumannii ATCC 17978 and Pseudomonas aeruginosa ATCC 27853 were used as the reference strains. Susceptibility screening tests were done against ceftazidime (30 μg), trimethoprim sulfamethoxazole (1.25/23.75 μg), cefepime (30 μg), amikacin (30 μg), cefotaxime (30 μg), ciprofloxacin (5 μg), piperacillin/tazobactam (100/10 μg), doxycycline (30 μg), cefotaxime (30 μg), imipenem (10 μg), meropenem (10 μg), tetracycline (30 μg), and gentamicin (10 μg).
All clinical isolates of Acinetobacter were tested for the presence of Ade B gene by real-time PCR. The PureLink RNA Mini Kit (supplied by Thermo Fisher Scientific, Ambion RWhatman, USA) is used for RNA extraction. After an overnight pure growth on MacConkey, 2–3 bacterial colonies were dissolved in 1000 μl of Nutrient Broth and left for overnight incubation at 37°C. RNA extraction was performed according to instructions of the manufacturer. Total RNA from all isolates was reverse transcripted into complementary DNA (cDNA) using cDNAkit (supplied by Thermo Fisher Scientific).
According to manufacturer's protocol, the reagents of the kit were combined to form 2X RT Master Mix, to which an equal volume of RNA sample was added. Real-time PCR was performed on 7500 Real Time Fast PCR instrument (Applied Biosystem), using Syber Green with the following primers: forward primer 5'-GGATTATGGCGACAGAAGGA-3' reverse primer, 5'-AATACTGCCGCCAATACCAG-3' (supplied by Thermos Fisher Scientific). Relative expression levels of tested genes were calculated according to the expression of the 16 S ribosomal RNA (rRNA) housekeeping gene of Acinetobacter. Amplification was designed including 12.5 μl of SYBR Green Qpcr Master Mix, 0.3 μl of primers, less than or equal to 500 ng cDNA, and nuclease-free water to 25 μl. The reaction conditions were 95°C for 10 min for the first denaturation, 95°C for 15 s for denaturation, 60°C for one minute for annealing and extension for 40 cycles, and melt curve at 72–95°C.
This study was approved by the Research Ethics Committee. Written informed consent was obtained from all participants or their relatives.
Data were fed to the computer and analyzed using IBM SPSS software package version 20.0. (SPSS Inc., Chicago, Illinois, USA) Qualitative data were described using number and percentage. The Kolmogorov–Smirnov test was used to verify the normality of distribution. Quantitative data were described using range (minimum and maximum), mean, SD, and median. Significance of the obtained results was judged at the 5% level. Kruskal–Wallis test is used to compare between more than two studied groups for abnormally distributed quantitative variables.
| Results|| |
This study was carried out in the period between October 2016 and October 2018. It was done at Clinical Pathology Department, Faculty of Medicine, Menoufia University Hospitals. The patients were selected from ICU of Menoufia University. A total of 70 Acinetobacter isolates were obtained from 614 clinical samples. The prevalence of Acinetobacter among the clinical isolates was 11.4%.
As shown in [Table 1], ∼87.1% had associated co-morbidity, and 72.9% of patients infected with Acinetobacter were exposed to invasive procedure. The incidence of Acinetobacter infection was high in patients with duration more than 7 days in hospital (71.4%). Moreover, 55.7% of patients infected with Acinetobacter were exposed to previous antibiotic therapy.
|Table 1 Demographic and clinical characteristics of Acinetobacter isolate cases|
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The highest isolation of Acinetobacter was from respiratory samples (sputum, endotracheal aspirate, and bronchial aspirate) 64.3%, followed by blood (27.1%), urine (5.7%), and wound swab (2.9%), as shown in [Table 2].
|Table 2: Distribution of isolated Acinetobacter according to clinical samples|
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Regarding antibiotic susceptibility, 17.1% were susceptible to all antibiotics, 14.3% were susceptible to one antibiotic, and 7.1% were susceptible to two antibiotics, whereas Multidrug resistant (MDR) cases were represented by 5.7% and extensive drug resistance was represented by 55.7% of the clinical isolates, as shown in [Table 3].
|Table 3: Distribution of the Acinetobacter cases regarding AB antibiotic resistance|
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Regarding antimicrobial susceptibility by disk diffusion method, Acinetobacter isolates were resistant to ceftazidime (51%), trimethoprim sulfamethoxazole (50%), cefepime, amikacin, CTX, ciprofloxacin, piperacillin/tazobactam, doxycycline (43% each), cefotaxime (42%), imipenem and meropenem (41%), tetracycline (40%), and gentamicin (36%), as shown in [Table 4].
On comparing Ade B gene expression, there was a highly significant difference between both MDR and Extreme drug resistant (XDR) and susceptible isolates (P < 0.001) and also between both MDR and XDR and isolates resistant to one or antibiotics, whereas there was no significant difference between susceptible isolates and isolates resistant to one or antibiotics (P = 0.22), as shown in [Table 5].
|Table 5: Comparison between different antibiotic resistance patterns of Acinetobacter isolates regarding gene expression|
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| Discussion|| |
Over the past decades, Acinetobacter infections have grown from a limited problem to a major cause of hospital-acquired infections worldwide. It has transformed from a monodrug-resistant to a multidrug-resistant or pandrug-resistant organism. The resistant mechanisms include formation of inactivated enzymes, gene mutations in chromosomes, changes in outer membrane porins, and the active drug efflux mechanism. The Ade ABC efflux pump consists of Ade A (membrane fusion), Ade B (multidrug transporter), and Ade C (outer membrane) genes.. The objective of the present study was to determine the role of the Ade B gene in multidrug-resistant Acinetobacter isolated from ICU of Menoufia University Hospitals. From 614 hospitalized patients admitted to ICUs of Menoufia University Hospitals, 70 Acinetobacter isolates were obtained (11.4%). This was in agreement with other study performed by Fouad et al. in Cairo, which found that Acinetobacter isolates represented 10% of the total isolates. A lower prevalence rate was observed by Anuradha et al. in India (3%), whereas a higher prevalence rate was detected by Uwingabiye et al. (24.8%) and Banergee et al. in India (42.9%).
This study revealed that most of the Acinetobacter-infected patients in ICUs of Menoufia University Hospitals had co-morbidities such as chronic liver diseases, chronic lung diseases, diabetes, and hypertension (87.1%); patients were under corticosteroids therapy; patients were exposed to invasive procedures (72.9%); most patients were staying in the hospitals for more than 7 days (71.4%); and patients had received previous antibiotic regimen (55.7%). In agreement with these results, García-Garmendia et al.reported that immunosuppression, unscheduled admission, respiratory failure at admission, previous antimicrobial therapy, previous sepsis in ICU, invasive procedures, and length of hospital stay were independently related to infection by multidrug-resistant Acinetobacter. Therefore, rational antibiotic use is an important risk management strategy used to prevent and reduce opportunistic pathogen infections in hospitals.
In the present study, the clinical Acinetobacter isolates were most commonly isolated from respiratory samples (64.3%) followed by blood (27.1%), urine samples (5.7%), and lastly, pus and burn wounds (2.9%). These results coincide with Amudhanet al. in India, who reported similar results. Thus, more attention is required when monitoring the respirator and the nursing staff to prevent respiratory tract infections in ICU. On the contrary, Sharma et al., in India, reported that maximum Acinetobacter isolates were from pus (41.5%) followed by respiratory secretions (28%). In our study 61.4% of clinical isolates of Acinetobacter were MDR or XDR, and this coincides with the results of Joshi et al., who found that 62% of Acinetobacter isolated from ICU of a Tertiary Care Hospital, Varanasi, India, were MDR and XDR. In another retrospective audit by Khan et al in an ICU, MDR Acinetobacter isolates were 70%. The resistant mechanisms of Acinetobacter include many pathways. The efflux system is a mechanism of antibiotic resistance involving the extrusion of toxic substrates from within cells into the external environment. The Ade ABC efflux pump belongs to the RND efflux system in Acinetobacter, which consists of Ade A (membrane fusion), Ade B (multidrug transporter), and Ade C (outer membrane) genes. In this study, the role of the efflux pump Ade B gene among the Acinetobacter isolates was determined, and we found that there was significant increase in Ade B gene expressed in MDR and XDR isolates in relation to susceptible isolates. This coincides with the results of Jassim et al. and also the same results were approved by Nejad et al., RastegarLari, and Magnet et al.. However, in a study performed by Bratu and colleagues, there was no correlation between Ade B gene expression and resistance to aminoglycosides, fluoroquinolone, or β-Lactamases. This may be because the resistance mechanisms have regional differences, which may be caused by a different phenotype or genotype of the clinically collected strains.
| Conclusion|| |
Ade B gene plays a vital role in multidrug resistance in clinical Acinetobacter isolates. These results may benefit to design active efflux pump inhibitors. Moreover, implementation of strict microbial policies and infection control programs may prevent the rapid dissemination of this organism.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]