Home About us Editorial board Search Ahead of print Current issue Archives Submit article Instructions Subscribe Contacts Login 


 
 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 33  |  Issue : 1  |  Page : 205-209

Role of AdeB gene in multidrug-resistance Acinetobacter


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 Submission31-Dec-2018
Date of Decision17-Jan-2019
Date of Acceptance10-Feb-2019
Date of Web Publication25-Mar-2020

Correspondence Address:
Reem M ElKholy
ShebinElkom, Faculty of Medicine, Yaseen Abdelghafar Street 32511
Egypt
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_434_18

Rights and Permissions
  Abstract 

Objective
The aim was to determine the role of the Ade B gene in multidrug-resistant Acinetobacter isolated from ICUs of Menoufia University Hospitals.
Background
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.
Results
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.
Conclusion
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 Top


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[1].

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[2].

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[3].

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[4]. 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 Top


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.

Statistical analysis

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 Top


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

Click here to view


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

Click here to view


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

Click here to view


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].
Table 4: Antimicrobial susceptibility pattern of Acinetobacter isolates

Click here to view


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

Click here to view



  Discussion Top


Over the past decades, Acinetobacter infections have grown from a limited problem to a major cause of hospital-acquired infections worldwide[5]. 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.[4]. 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.[6] 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%)[7], whereas a higher prevalence rate was detected by Uwingabiye et al. (24.8%)[8] and Banergee et al. in India (42.9%)[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.[10]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.[11] 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%)[12]. In our study 61.4% of clinical isolates of Acinetobacter were MDR or XDR, and this coincides with the results of Joshi et al.[13], 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[14] in an ICU, MDR Acinetobacter isolates were 70%. The resistant mechanisms of Acinetobacter include many pathways[14]. The efflux system is a mechanism of antibiotic resistance involving the extrusion of toxic substrates from within cells into the external environment[2]. 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[2]. 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.[2] and also the same results were approved by Nejad et al.[15], RastegarLari[16], and Magnet et al.[17]. 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[18]. 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 Top


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

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Parisa M, AiMitra S, Farzaneh H, Fatemeh F. Distribution and expression of efflux pump gene and antibiotic resistance in Acinetobacterbaumannii. Clin Microb Infect 2018; 10:5812.  Back to cited text no. 1
    
2.
Jassim KA, Kassim K, Shurook MK. AdeABC efflux pump genes in multidrug resistant Acinetobacterbaumannii isolates. Clin Microb Infect 2016; 10:17795.  Back to cited text no. 2
    
3.
Moustafa NT, Ahmed O, Amal E, Wael M. Effect of efflux pump inhibitor carbonyl cyanide3-chlorophenylhydrazone on the minimum inhibitory concentrationof aminoglycosides in Acinetobacterbaumannii clinical isolates. Jundishapur J Microbiol 2018; 10:5812.  Back to cited text no. 3
    
4.
Wong EW, MohdYusof MY, Anbazhagan D, Ong SY, Sekaran S. Disruption of AdeBgene has a greater effect on resistance to meropenemsthanAdeAgene in Acinetobacterspp.isolated from University Malaya Medical Centre. Singapore Med 2009; 50:822.  Back to cited text no. 4
    
5.
Marcella A, Michael K. Acinetobacterbaumannii: an emerging and important pathogen. Jclin 2010; 17:363–369.  Back to cited text no. 5
    
6.
Fouad M, Attia SA, Tawakkol MW, Hashem MA. Emergence ofcarbapenem-resistant Acinetobacterbaumannii harboring the OXA-23 gene inintensive care units of Egyptian hospitals. Int J Inf Dis 2013; 17:1775.  Back to cited text no. 6
    
7.
Baker AM, Makled AF, Salem EH, Salama AA, Ajlan SE. Phenotypic and molecular characterization of clinical Acinetobacter isolates from Menoufia University Hospitals. Menoufia Medical Journal 2017; 30:1030.  Back to cited text no. 7
    
8.
Uwingabiye J, Mohammed F, Abdelhay L, Adil M. Acinetobacter infectionsprevalencand frequency of the antibiotics resistance: comparative study of intensive care units vs other hospital units. Pan Afr Med J 2016; 10:116–204.  Back to cited text no. 8
    
9.
Banerjee T, AnwitaM, Arghya D, Swati S. High prevalence and endemicity of multidrug resistant Acinetobacter spp. in Intensive Care Unit of a Tertiary Care Hospital, Varanasi, India. Journal of Pathogens 2018; 10:1155.  Back to cited text no. 9
    
10.
García-Garmendia JL, Ortiz-Leyba C, Garnacho-Montero J, Jiménez-Jiménez FJ, Pérez-Paredes C, Barrero-Almodóvar AE, et al. Risk factors for Acinetobacterbaumannii nosocomial bacteremia in critically ill patients: a cohort study. Clin Infect Dis 2001; 10:1086.  Back to cited text no. 10
    
11.
Amudhan SM, Sekar U, Arunagiri K, Sekar B. OXA beta-lactamasemediatedcarbapenem resistance in Acinetobacter baumannii. J Med Microbiol 2011; 29:269–274.  Back to cited text no. 11
    
12.
Sharma P, Bashir UY, Kaur S, Kaur P, Aggarwa A. Emerging antimicrobial resistance and clinical relevance of Acinetobacter isolates in a tertiary care hospital of rural area of Punjab, India. J Micro Anti Agents 2015; 1:8–12.  Back to cited text no. 12
    
13.
Joshi GS, Litake MG. Acinetobacter baumannii: an emergingpathogenic threat to public health. J Clin Infect Dis 2015; 3:25–36.  Back to cited text no. 13
    
14.
Mathai AS, Oberoi A, Madhavan S, Kaur P. Acinetobacter infections in a tertiary level intensive care unit in northern India: epidemiology, clinical profiles and outcomes. J Infect Public Health 2012; 5:145–152.  Back to cited text no. 14
    
15.
JaponiNejad AR, Sofian M, Ghaznavi-Rad E. Molecular detection of AdeABC efflux pump genes in clinical isolates of Acinetobacterbaumannii and their contribution in imipenem resistance. Iran South Med J 2014; 17:815–823.  Back to cited text no. 15
    
16.
Maryam B, Malihe T, Abdollah A, Abbas B, AbdolazizRastegar L. Detection of AdeABC efflux pump genes in tetracycline-resistant Acinetobacterbaumannii isolates from burn and ventilator-associated pneumonia patients. J Pharm Bioallid 2014; 6:229–232.  Back to cited text no. 16
    
17.
Rastegar L, Abdollah A, Ali H. AdeR-AdeS mutations & overexpression of the AdeABC efflux system in ciprofloxacin-resistant Acinetobacter baumannii clinical isolates. Indian J Med Res 2018; 10:4103.  Back to cited text no. 17
    
18.
Simona B, David L, Don Antonio M, Claudiu G, John Q. Correlation of antimicrobial resistance with β-lactamases, the OmpA-like porin, and efflux pumps in clinical isolates of Acinetobacter baumannii endemic to New York City. Antimicrob Agents Chemother 2008; 10:1128.  Back to cited text no. 18
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

Top
 
 
  Search
 
Similar in PUBMED
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
Abstract
Introduction
Materials and Me...
Results
Discussion
Conclusion
References
Article Tables

 Article Access Statistics
    Viewed35    
    Printed0    
    Emailed0    
    PDF Downloaded4    
    Comments [Add]    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]