Skip to main content
Download PDF

Detection of AdeAB, TetA, and TetB efflux pump genes in clinical isolates of tetracycline-resistant Acinetobacter baumannii from patients of Suez Canal University Hospitals

BMC Microbiology volume 25, Article number: 63 (2025) Cite this article

Abstract

Background

Acinetobacter baumannii is an opportunistic bacteria associated primarily with hospital-acquired infections. Its tendency to acquire or donate resistance genes to neighboring bacteria is a major concern. Tetracyclines have shown promise in treating A. baumannii infections, but tetracycline resistance is growing globally in A. baumannii isolates.

Objectives

The study aimed to study (1) the prevalence of multidrug-resistant (MDR) A. baumannii infections at Suez Canal University Hospitals, (2) the distribution of efflux pump genes AdeA &B, TetA, and TetB, and (3) the effect of efflux pump inhibitor (CCCP) on tetracycline-resistant isolates.

Methods

Clinical samples (457) were collected (blood, urine, sputum, ETA, pus, and pleural fluid), followed by A. baumannii isolation and identification, PCR detection of efflux pump genes, and detection of tetracycline susceptibility and its MIC before and after treatment with the efflux pump inhibitor (CCCP).

Results

A total of 31 A. baumannii isolates were recovered (6.78%). The highest rate of isolation was from the ICU (48.3%) from the ET aspirate samples (48.3%). The efflux system AdeA and TetB genes were distributed in 100% of isolates, whereas AdeB was found in 93.5% of isolates and the TetA gene in 87.1% of isolates. All A. baumannii isolates were MDR showing resistance to three or more classes of antibiotics. 45% of the isolates showed a 4-fold reduction of MIC and 12.9% showed a 2-fold reduction in the MIC.

Conclusions

Efflux pump is an important mechanism for tetracycline resistance among A. baumannii isolates.

Peer Review reports

Introduction

Acinetobacter baumannii (A. baumannii), an aerobic, pleomorphic, non-motile, gram-negative, coccobacilli, is an opportunistic bacterial pathogen primarily associated with hospital-acquired infections (nosocomial), particularly among immunocompromised individuals causing bacteremia, urinary tract infection, ventilator-associated pneumonia (VAP), and wound infections. Sporadic cases of peritonitis, endocarditis, meningitis, osteomyelitis, and arthritis have also been reported [1]. VAP is the most frequent ICU-acquired infection, occurring in 9 to 24% of patients intubated for longer than 48 h. Colonization of the digestive tract in intensive care unit patients is an important epidemiologic reservoir for multi-drug-resistant A. baumannii infections in hospital outbreaks [2]. A. baumannii naturally encodes efflux pumps, providing it with intrinsic resistance to antibiotics beside low membrane permeability, both of which allow it to survive a variety of antibiotics used, making it challenging to treat in some clinical settings, particularly with critically ill patients. Thus, there are now just a few antibiotics projected to be effective against the severe types of A. baumannii infections leading to the augmentation of fatality rates and healthcare expenditures [3].

Unfortunately, several methods, including an efflux pump, a ribosome protection system, and enzyme modification, can lead to bacterial antibiotic resistance. A. baumannii-resistant strains continue to appear and spread, and there aren’t many effective treatment choices [4]. According to an Egyptian study, 61.4% of the Acinetobacter spp. isolates were found to be multi- and extensive drug-resistant bacteria [5]. For this reason, alternative antibiotics have been studied for use in clinical settings. Tetracyclines, such as doxycycline and minocycline, have demonstrated encouraging microbiological and clinical efficacy in the treatment of A. baumannii infections. It has been found that tetracyclines can be successfully used in conjunction with other antibiotics to treat 87.5% of bloodstream infections and 71.9% of respiratory infections [6].

The efflux pumps are involved in clinically relevant resistance to antimicrobial agents. They are responsible for reducing antibiotic accumulation via transporting antibiotics outside the bacterial cells [7]. There are five families of efflux-pump proteins that are associated with multidrug resistance (MDR) in bacteria, based on amino acid sequence homology, (1) the “multidrug and toxic compound extrusion” family (MATE), (2) the “adenosine triphosphate (ATP)-binding cassette” superfamily (ABC), (3) the “small multidrug resistance” family (SMR), (4) the “major facilitator superfamily (MFS), and (5) the “resistance-nodulation-cell division” family (RND). Drug efflux pumps are found in both gram-negative and gram-positive bacteria, with more complexity in Gram-negative ones due to the molecular architecture of the cell envelope (reduced drug intake) and efflux pumps (active drug export). A. baumannii antimicrobial resistance has been linked to more than one class of efflux pumps: the SMR family, the MATE family, and the RND family [8]. The tetracycline-specific efflux pump system in A. baumannii is the synergistic act of TetA and TetB, members of the MFS superfamily efflux pump, and AdeABC, members of the RND superfamily efflux pump. TetA efflux provides resistance against tetracycline, but not against minocycline or doxycycline, while TetB efflux provides resistance against tigecycline, but not against tetracycline and minocycline. Additionally, it has been discovered that TetA functions in concert with AdeABC to provide a crucial tigecycline resistance mechanism [9].

Since efflux pumps have a significant role in the resistance mechanisms of A. baumannii, efflux pump inhibitors (EPIs) have been extensively investigated to confer one potential solution to combat antibiotic resistance by blocking their efflux pumps via “competitive or non-competitive inhibition”, “dissipation” of the proton gradient needed by efflux pumps to export antibiotics, “suppression” of the expression of genes that encode efflux pumps, “blocking the inner or outer membrane protein” used for efflux, “disruption” of the efflux pump assembly, and “changing the structure of the medication” so that it cannot be recognized [10]. One of these inhibitors is carbonyl cyanide 3-chlorophenylhydrazone (CCCP), an oxidative phosphorylation uncoupler that affects bacterial membrane ionic gradients. This compound has been efficiently utilized in combination with antibiotics to increase their susceptibility [11].

Given the high prevalence of multidrug-resistant (MDR) A. baumannii infections at Suez Canal University Hospitals, this study aimed to investigate the distribution of AdeAB, TetA, and TetB genes and the effect of efflux pump inhibitor (CCCP) on tetracycline-resistant isolates among isolates from patients in this institution.

Materials and methods

Study population and design

This observational cross-sectional descriptive study was conducted at the Suez Canal University Hospitals in Ismailia, Egypt, from September 2022 to September 2023. Samples were collected from different intensive care units (ICUs) and other hospital wards and were processed in the Medical Microbiology and Immunology Department laboratory, Faculty of Medicine, Suez Canal University.

Ethical considerations

The current study was implemented in coordination with the guidelines of the Declaration of Helsinki. Ethical approval was gained from the Research Ethics Committee of the Faculty of Medicine, Suez Canal University, Egypt, # 5024. Informed consent was obtained from the patients, which addressed all the steps of the study and their right to withdraw at any time.

Inclusion criteria

Patients of both sexes (male & female) and all age groups who showed signs and symptoms of infections and agreed to participate were included in this study. Samples included were urine from catheterized and non-catheterized patients, respiratory specimens from intubated and non-intubated patients, Pus and wound swabs, pleural fluid, and blood.

Exclusion criteria

Patients who received antibiotic treatment in the last 48 h or refused to participate were excluded.

Study procedure

Full history taking, and thorough physical examination

All enrolled patients were asked and examined for underlying medical conditions or certain types of healthcare exposures, such as:

  • Immunocompromising conditions.

  • Recent frequent or prolonged stays in health care settings.

  • Invasive medical devices such as breathing tubes, feeding tubes, and central venous catheters.

  • Open wounds.

  • A history of taking certain antibiotics for long periods.

For every patient admitted to internal wards or intensive care units, the following data was found age, length of stay, number of days needed for mechanical ventilation, and survival until hospital discharge. Additionally, microbiological results were obtained to identify A. baumannii-positive cultures.

Samples collection and preservation

Various samples were collected throughout the study for presumptive A. baumannii isolation and identification according to standard laboratory procedures, including culture on Enriched and selective agar media and incubation under appropriate conditions following the guidelines provided by the Clinical and Laboratory Standards Institute (CLSI) [12] and the World Health Organization (WHO) [13]. We followed the CDC’s recommendations to prevent the spread of drug-resistant Acinetobacter spp. These included implementing contact isolation precautions, enhancing environmental cleaning, using dedicated patient care equipment, and ensuring the prudent use of antibiotics. Additionally, healthcare personnel adhered to strict infection control practices, such as wearing gowns and gloves when entering patient rooms and maintaining rigorous hand hygiene protocols [14].

a. blood samples

Blood samples were taken before the onset of antimicrobial therapy and throughout the feverish phase when feasible. Ten milliliters of blood were extracted and promptly injected into sterile blood culture bottles holding fifty milliliters of brain heart infusion (BHI) broth after the skin had been cleaned and disinfected with 70% alcohol. Two milliliters of blood were taken from neonatal patients and placed into sterile culture flasks with ten milliliters of BHI broth. Culture flasks were brought straight to the lab and kept there between 35 and 37 °C.

b. urine samples

Urine samples were taken midstream in non-catheterized patients and placed in a sterile wide-mouthed container. In catheterized patients, their catheters had clamped off. After cleaning the needle puncture site and inserting the syringe at a 45° angle, urine was aspirated using a syringe. Urine specimens were rapidly transported to the laboratory (less than two hours post-voiding). Urine samples were refrigerated at 4 °C until processing if a delay was expected.

c. Sputum samples

Early morning samples were collected from conscious patients with lower respiratory tract infections. Patients were instructed to gargle with water immediately before collecting samples to reduce the number of contaminating oropharyngeal bacteria. Sputum was collected by asking patients to cough deeply into a sterile wide-mouthed container with a tightly fitted cap. Only thick sputum secretions from the lung were acceptable.

d. endotracheal aspirate samples (ETA)

A sterile silicon tracheal catheter was used to collect ETA. It was inserted through the trachea until resistance (the level of the trachea’s carina) was met, at this point it was retracted about 2 cm. The vacuum was then released, and the catheter was carefully removed using rotating motions. Following the catheter’s withdrawal, the exudates were flushed into a sterile container using a sterile syringe and 2 mL of sterile 0.9% normal saline. After that, the samples were delivered to the microbiology lab in a maximum of two hours.

e. pus and wound exudate samples

The traumatized region was cleaned with sterile saline to remove contaminated materials like slough, necrotic tissue, or dry exudates. Pus and wound exudates were either collected by inserting a sterile cotton swab deeply down the leading edge of the wound or aspirated using a syringe. The specimens were immediately brought to the lab in Amie’s transport medium.

f. pleural fluid specimens

The pleural fluid specimen was aseptically obtained using thoracocentesis and placed in a tube containing a sterile anticoagulant (ethylenediaminetetraacetic acid, or EDTA) before being sent right away to the lab.

Bacterial isolation and identification

For bacterial isolation, all collected samples were inoculated into blood agar and MacConkey agar media (Oxoid, UK) and incubated aerobically at 35 ± 2˚C for 24 h. For initial identification of the isolates, they underwent a hanging drop test, and biochemical tests including “catalase, citrate, oxidase, coagulase, oxidative fermentation, indole, Methyl Red (MR), Voges-Proskauer (VP), urease, H2S production, gelatin hydrolysis, and bile solubility”, and growth at temperatures of 37 °C and 44 °C.

Molecular confirmation of A. baumannii isolates (genotypic characteristic) was performed using conventional PCR amplification of the “BlaOXA−51−like gene”. DNA of the isolates was extracted using a DNA extraction kit according to the manufacturer’s instructions. DNA extraction kit was supplied from “QIAGEN Company (Cat. No.27200-4)”. DNA concentration and purity were measured using a spectrophotometer. The PCR mixture was done in a total volume of 25 µl including 1 µl MgCl2 (1.5 mM), 0.3 µl Taq DNA polymerase (500 U), 2.5 µl 10x PCR buffer, 0.5 µl dNTP (200 µM), 1 µl of each primer) 10 pmol/ml) and 2 µl of DNA template (5 ng genomic DNA). The primer sets “F: 5’-TAATGCTTTGATCGGCCTTG-3’ and R: 5’-TGGATTGCACTTCATCTTGG-3’” were used for amplification of the “OXA-51-like” gene [15]. The amplification conditions: initial denaturation at 94 °C for 5 min, and 30 cycles of 94 °C for 45 s, 52 °C for 40 s, 72 °C for 45 s, and a final extension at 72 °C for 6 min. Amplified fragments were separated by electrophoresis in 2% agarose gel at 5 volts/cm for 2 h. Finally, the fragments of the BlaOXA-51-like gene with 353 bp size were stained with ethidium bromide and detected under a UV transilluminator documentation system [16].

Molecular detection of Efflux pump genes (AdeA, AdeB, TetA, and TetB)

Multi-drug-resistant A. baumannii isolates were screened for four Efflux pump genes (AdeA, AdeB, TetA, and TetB). They were tested by conventional PCR using the primers illustrated in Table 1. PCR cycling conditions included initial denaturation at 94 °C for 5 min, and 30 cycles of 94 °C for 45 s, 56–58 °C for 40 s, 72 °C for 45 s, and a final extension at 72 °C for 6 min. Amplified fragments were separated by electrophoresis in 2% agarose gel at 5 volts/cm for 2 h.

Table 1 Gene-specific primers used to detect efflux pump genes in A. Baumannii isolates
Full size table

Antimicrobial sensitivity testing

a. standard disk diffusion method

Phenotypic detection of antibiotic resistance was done using the “Kirby-Bauer disk diffusion method” on Mueller Hinton agar and incubated at 35ºC for 16–18 h according to Clinical and Laboratory Standard Institute guidelines of CLSI, 2022, using A. baumannii ATCC19606 as a reference strain [17]. A. baumannii isolate ATCC 19,606 was recovered in the US before 1948. It has been used as a reference and model organism in many studies involving antibiotic resistance and pathogenesis of A. baumannii. The Acinetobacter baumannii ATCC 19,606 strain is known to harbor efflux pump genes such as TetA and AdeABC, which mediate resistance to tetracycline. The expression of the AdeABC efflux pump genes is tightly regulated by the AdeRS two-component system in A. baumannii ATCC 19,606. Both amino acid substitutions and transposon insertion have been shown to continuously turn on the AdeRS two-component system and then constitutively activate AdeABC efflux pump gene expression in clinical isolates or laboratory mutants [18]. The MIC of tetracycline for this strain, with a value of 6 µg/ml to tetracyclines [19]. Inhibition of A. baumannii efflux pump genes by CCCP inhibitor restores its antimicrobial susceptibility [20].

The following antibiotic disks were used: “Cefepime (30µg), Ceftazidime (30µg), Meropenem (10µg), Gentamycin (10µg), Amikacin (30µg), Ciprofloxacin (5µg), Levofloxacin (5µg), Tetracycline (30µg), and Doxycycline (30µg)”. Bacterial isolates that are resistant to at least one agent in at least three classes of antibiotics are classified as multidrug-resistant (MDR), while isolates that are responsive to just one or two antimicrobial classes are classified as extensively drug-resistant (XDR) [21].

b. minimal inhibitory concentration determination

The agar dilution method was used to assess the minimal inhibitory concentration (MIC) of tetracycline both before and after treatment with the efflux pump inhibitor “carbonylcyanide 3-chlorophenylhydrazone” (CCCP) (Sigma-Aldrich, Dorset, United Kingdom) [22].

CCCP was added to Mueller-Hinton (M-H) agar plates, leading to increased intracellular concentration of the antibiotic reducing the MIC in isolates with active efflux pumps. M-H agar plates were prepared with 0.5 to 1024 µg/mL concentration of tetracycline and CCCP (25 µg/mL) was added to each. Then, the MIC of tetracycline was determined for all tetracycline-resistant isolates against the A. baumannii isolate. M-H agar plates with CCCP without the antibiotic were used as controls. The effect of the efflux pump inhibitor was determined by detecting a ≥ 4-fold increase in the susceptibility after treatment with CCCP [23].

Statistical analysis

Data collected will be reviewed, coded, and statistically analyzed using Statistical Package for the Social Science (SPSS) program version 28 (Inc, Chicago, Illinois, USA). Data presentation was performed via tables and graphs. Qualitative data were presented as numbers and percentages while quantitative data were presented as mean ± Standard Deviation. Fisher’s exact tests were used for qualitative variables. The McNemar test was used to compare tetracycline resistance pre- versus post-adding CCCP. Also, the Wilcoxon signed-rank test was used to compare MIC scores. A p-value of < 0.05 was considered statistically significant.

Results

Isolation, identification, and phenotypic detection of A. Baumannii isolates

A total of 457 examined clinical samples were analyzed for bacterial isolation. A total of 31 A. baumannii isolates were recovered (6.78%). Other organisms represented 426 samples (93.3%) of the isolated microorganisms.

The ages of the patients ranged from 13 days old to 72 years old. A. baumannii isolates were obtained more frequently from males than females (Table 2).

Table 2 Frequency distribution of patients infected with A. Baumannii according to gender and age (N = 31)
Full size table

The highest rate of A. baumannii isolation was from the ICU (48.3%), while the lowest rate was from the urology and orthopedics departments (3.3%, each) (Table 3). The highest rate of A. baumannii isolation was from the ET aspirate samples (48.3%), while the lowest rate was from wound swabs (3.2%) (Table 4).

Table 3 Frequency distribution of the isolated MDR A. Baumannii isolates according to the hospital wards from which the sample was obtained (N = 31)
Full size table
Table 4 Frequency distribution of the isolated MDR A. baumannii strains according to the type of the samples (N = 31)
Full size table

Preliminary isolation and identification of Acinetobacter spp. based on Gram-negative staining and colony morphology. A. baumannii are Gram -ve, non-motile coccobacilli. Macroscopic characterization of colonies on blood agar and MacConkey agar showed small smooth, opaque, raised, and creamy colonies on blood agar, and pure purple or mucoid colonies on MacConkey agar. Biochemical tests used to identify the A. baumannii isolates were reported in Table 5.

Table 5 Biochemical tests are used to identify A. Baumannii isolates
Full size table

Genotypic detection of A. Baumannii isolates and efflux pumps genes

The 31 isolates (100%) were confirmed to be A. baumannii by conventional PCR detection of the BlaOXA−51−like gene by gel electrophoresis at 353 bp (Fig. 1). AdeA and TetB genes were detected in 100% of isolates (31 isolates), where AdeB was detected in 93.5% of isolates (29 isolates) and TetA gene in 87.1% of isolates (27 isolates). Detection of the genes AdeA, AdeB, TetA, and TetB by gel electrophoresis were at 106 bp, 185 bp, 210 bp, and 586 bp respectively (Fig. 2A-D).

Fig. 1
figure 1

Detection of BlaOXA−51−like gene by agarose gel electrophoresis. Lane M shows a 100 bp molecular strand DNA ladder. Lane 1–16 shows positive samples (353 bp)

Full size image
Fig. 2
figure 2

Detection of efflux pump genes by agarose gel electrophoresis. Lane M shows a 100 bp molecular strand DNA ladder, A: AdeA gene; Lane 1–16 shows positive samples (106 bp), B: AdeB gene; Lane 1–16 shows positive samples (185 bp), C: TetA gene; Lane 9, 10,11 and 12 show positive specimens (210 bp), D: TetB gene; Lanes 1–14 shows positive specimens (586 bp)

Full size image

Antibiotic susceptibility test

An antibiotic susceptibility test was done on the 31 A. baumannii isolates by using the disk diffusion method (Kirby Bauer method). It showed highest resistance to ceftazidime (30 isolates, 96.8%) followed by cefepime (28 isolates, 90.3%), levofloxacin and ciprofloxacin (27 isolates, 87.1%), amikacin (26 isolates, 83.9%), gentamicin and meropenem (23 isolates, 74.2%) and tetracycline (22 isolates, 71%). The least resistance was to doxycycline (18 isolates, 58.1%) (Fig. 3).

Fig. 3
figure 3

Antibiotic susceptibility pattern of the studied MDR A. baumannii isolates by the disc diffusion method. The figure shows the percentage of resistance to antibiotics

Full size image

The minimal inhibitory concentration of tetracycline

Minimal inhibitory concentration (MIC) was done using the agar dilution method, which showed 67.7% (21 isolates) resistance to tetracycline (Fig. 4). Also, the efflux pump inhibitor carbonylcyanide3-chlorophenylhydrazone (CCCP) determined the MIC of tetracycline after treatment. 45% of the isolates (14 isolates) showed a reduction of MIC 4 folds or more, and 12.9% (4 isolates) showed a 2-fold reduction in the MIC (Table 6; Fig. 5). By comparing the tetracycline resistance before and after adding CCCP, it showed a significant difference (p < 0.05), as the percentage of tetracycline resistance changed from 67.7% (before CCCP treatment) to 35.5% (after CCCP treatment) (Table 7). AdeA and TetB genes were detected in 100% of both resistant (21 isolates) and sensitive isolates (10 isolates). In comparison, AdeB was detected in 95.2% (20 isolates) of resistant and 90% (9 isolates) of sensitive isolates and TetA gene was detected in 85.7% (18 isolates) of resistant and 90% (9 isolates) of sensitive isolates (Table 8).

Fig. 4
figure 4

Distribution of tetracycline susceptibility by agar dilution method. The figure shows that 67.7% of isolates were resistant to tetracycline

Full size image
Table 6 MIC of the samples before and after adding CCCP, showing the effectiveness of CCCP in reduction of the MIC
Full size table
Fig. 5
figure 5

The change of MIC of tetracycline after treatment by the efflux pump inhibitor carbonylcyanide3-chlorophenylhydrazone (CCCP)

Full size image
Table 7 Comparison of tetracycline resistance before and after adding CCCP
Full size table
Table 8 Distribution of AdeAB, TetA, and TetB genes among A. Baumannii isolates regarding MIC of samples (µg/ml)
Full size table

Discussion

Alarmingly, the MDR A. baumannii infection is spreading throughout the world. It is linked to several ailments in hospitalized patients, particularly in intensive care units. Because it is a pathogen with a high propensity to acquire and/or donate resistance genes to neighboring bacteria, it is currently on the priority lists of healthcare-associated organizations, including the World Health Organization (WHO), the National Institute of Health (NIH), and the Centers for Disease Control and Prevention (CDC). It has been identified as one of the six bacterial infections of greatest healthcare concern, ESKAPE (Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas, and Enterobacter) spp., with fatality rates in some patient populations reaching 80% [10]. Numerous virulence factors that have been found over time are thought to contribute to the pathogenesis of A. baumannii [3]. Physical elements like its hydrophobic surface; outer membrane structures like the outer membrane protein A (OmpA) [6]; lipopolysaccharides (LPS); and complex secretory apparatus like Type II [7] and Type VI [8] secretion systems, along with their substrates [9, 10] are among them. A. baumannii also possesses vital mechanisms for acquiring nutrients [11, 12]. These and other virulence factors work together to enable A. baumannii to create biofilms, colonize the host, elude the host’s defenses, and spread infection throughout the body’s organs [3, 13].

The study was carried out on 457 clinical samples, a total of 31 A. baumannii isolates were recovered (6.78%). The age of the patients ranged from 13 days old to 72 years old, with a percentage of (54.8%) from male patients and (45.2%) from female patients (Table 2). The MDR A. baumannii isolates were most recovered from the ET aspirates (48.3%) samples. The bacteria were also isolated from other clinical samples; urine (16.1%), sputum (10%), blood (10%), pleural effusion (6.2%), pus (6.2%), and wound swabs (3.2%) (Table 3). The most common sources of such isolates were from the ICUs (48.3%). The bacteria were also isolated from other hospital wards; Internal Medicine (16.2%), Surgery (9.7%), NICU (6.4%), Burn Unit (6.4%), PICU (6.4%), Urology (3.3%), and Orthopedics (3.3%) wards (Table 4).

The disparities in isolation rates across studies may be related to changes in the hospital setting, the quantity of specimens examined, and shifts in the clinical status of the patients. Studies from other hospitals in Egypt showed a low prevalence rate of A. baumannii. It was 20%, 16.1%, 11.4%, 10% in Benha [27], Ain Shams- Cairo [28], Menoufia [29], and Cairo [30] University Hospitals respectively. A lower prevalence rate was observed in India (3%) [31], whereas higher prevalence rates were also detected in India (24.8%) [32] and (42.9%) [33].

As demonstrated by the results of our study, in a Tertiary Care Hospital in Nepal, the majority (49.18%) were A. baumannii isolated from specimens related to the respiratory system, such as tracheal aspirate, bronchoalveolar lavage, and sputum. Similarly, ICU patients accounted for the greatest percentage of MDR isolates (60%), followed by surgical wards (22%) and medical wards (13%), with burn wards accounting for the lowest percentage (1%) [34]. When the study was repeated after 5 years, the results showed that the majority (47.2%) were isolated from specimens related to the respiratory system, such as tracheal aspirate, bronchoalveolar lavage, and sputum. Of all the MDR isolates, 58.3% came from male patients and 41.7% came from female patients. Similarly, ICU patients accounted for the greatest percentage of MDR isolates (49.6%), followed by surgical wards (19.9%) and medical wards (14.3%), with burn wards accounting for the lowest percentage (1.9%) [35]. In a study conducted in Hamad General Hospital, Qatar, the most commonly identified sites of A. baumannii infection were the respiratory tract (48.9%). Of all the MDR isolates, 76.2% came from male patients and 23.8% came from female patients. ICU patients accounted for the greatest percentage of MDR isolates (28.6%) [36].

In the present study, the blaOXA−51−like gene was used for the biological identification of A. baumannii. It is evident that the blaOXA−51−like gene is found in the great majority of A. baumannii isolates. Their detection could offer a quick and easy way to identify A. baumannii that would be more dependable than the most widely used biochemical identification method (Table 5) and easier to carry out than the current definitive methods, such as amplified rRNA gene restriction analysis if they are consistently found and unique to this species. A. baumannii is the most important species in terms of clinical implications, thus being able to quickly differentiate it from other members of the genus would be quite beneficial [37]. In our study, the 31 isolates (100%) were confirmed to be A. baumannii by conventional PCR detection of the blaOXA−51−like gene by gel electrophoresis at 353 bp (Fig. 1).

Previous studies also used the blaOXA−51−like gene for the detection of A. baumannii. It was proved that the blaOXA−51−like gene was the most prevalent gene carried by A. baumannii isolates. Previous studies in EGYPT demonstrated the provenance of the blaOXA−51−like gene in (95%) of Ain Sham-Cario University Hospitals isolates [28], (96%) of Assiut University Hospitals isolates [38], and (100%) of Kasr Al-Aini -Cairo Hospital [39]. Several studies conducted in the Middle East; in Yemen [40], Kuwait [41], Saudi Arabia [42], and Qatar [43], also confirmed the prevenance of the blaOXA−51−like gene in A. baumannii isolates.

Among the first β-lactamases found to be mediated by plasmids were the OXA β-lactamases. To date, more than 220 OXA-type-β-lactameases have been found. At first, they were confined to penicillins, but some of them developed the ability to confer resistance to cephalosporins. During the 1980s, isolates of carbapenem-resistant A. baumannii (CRAB) were revealed to exhibit plasmid-encoded β-lactamases, including OXA-23, OXA-40, OXA-58, and OXA-51. Ultimately, it was discovered that each strain of A. baumannii had a chromosomally encoded OXA-51, which was accountable for its antimicrobial resistance [44]. The blaOXA−23−like, blaOXA−24−like, blaOXA−51−like, blaOXA−58−like, and more recently, blaOXA−143−like are the five primary phylogenetic OXA-type subgroups that have been identified in CRAB [45]. According to reports, blaOXA−51 gene is inherent to A. baumannii and chromosomally encoded, whereas plasmids or chromosomes mediate blaOXA−23, blaOXA−24, and blaOXA−58 [44]. Hence, the blaOXA−51−like gene has been utilized as a marker for A. baumannii because every strain of the bacteria had a chromosomally encoded OXA β-lactamase (OXA-51-like). Nonetheless, non-A. baumannii species plus carbapenem-susceptible A. baumannii have been shown to harbor plasmids encoding OXA-51 [46].

In this study, the efflux system such as AdeAB, TetA, and TetB, was detected using the traditional PCR to detect various efflux pump genes, in recognition of their important mechanistic role in antibiotic resistance involving the extrusion of antimicrobials, such as tetracycline, from cells into the external environment [7]. Our research discovered AdeA and TetB genes in 100% of isolates, whereas AdeB was found in 93.5% of isolates and the TetA gene in 87.1% of isolates (Fig. 2A-D). Our findings suggest that the efflux pumps mediated by the TetA, TetB, and AdeAB genes play a crucial role in increasing resistance to various antibiotics, particularly tetracyclines.

Our findings match previous studies performed in EGYPT. The AdeA gene, which encodes one of the proteins that make up the tripartite system of the AdeABC efflux pump, was shown to be the most prevalent (82%) in clinical A. baumannii strains isolated from Benha University Hospital [47]. Comparable results were reported in Alexandria University Hospital, where most isolates harbored efflux pump encoding genes, as detected in our results, AdeA, AdeB, and other genes [48].

The results of our study are consistent with the results of studies around the world. The AdeA and AdeB genes were detected in 83.9% and 90.3% respectively of A. baumannii isolates recovered from an Iraqi hospital [49]. In a study from Iran, the prevalence of tetracycline resistance genes among tetracycline-resistant A. baumannii isolates was 99.2%, 86.7%, and 10% for AdeB, TetB, and TetA genes respectively [50]. In another study in Iran, the prevalence of the AdeB gene was 100% of the isolated A. baumannii [51]. In A. baumannii isolates recovered from patients in a teaching hospital in Jordan, the TetB gene is the most common (82.6%), while none of the isolates had the TetA gene [52]. The majority of the A. baumannii isolates recovered from Malaysian hospitals carried the AdeA gene (62.7%) [53]. Nemec et al in France reported the prevalence of the AdeA gene (81.9%) [54].

Different mechanisms of resistance to numerous antibiotic classes are displayed by A. baumannii. Efflux pumps are one of the resistance mechanisms found in A. baumannii. Antibiotics and other chemicals seep out of the bacteria due to these pumps, lowering the number of drugs in the bacterial cells [7]. The tetracycline-specific efflux pump system in A. baumannii comprises the collaborative function of TetA and TetB, which belong to the MFS superfamily of efflux pumps, alongside AdeABC, which is part of the RND superfamily of efflux pumps [9]. Several “Tet efflux pumps” that lead to resistance to tetracycline have been acquired by clinical isolates of A. baumannii. The TetA and TetB are the most prevalent, with “TetA efflux” conferring resistance to tetracycline but not to minocycline or doxycycline and “TetB efflux” conferring resistance to tetracycline and minocycline but not to tigecycline. It has also been found that “TetA efflux” acts synergically with the “AdeABC efflux” pumps to provide A. baumannii antimicrobial resistance [6]. The AdeABC efflux pump consists of three proteins; “AdeA (inner membrane fusion), AdeB (multidrug transmembrane transporter), and AdeC (outer membrane)”, which are chromosomally regulated by “adeS (sensor kinase) and adeR (response regulator)”. The two proteins work together to regulate efflux pump gene expression in response to environmental stimuli [47].

In the present study, all A. baumannii isolates were MDR showing resistance to three or more classes of antibiotics (Fig. 3). Bacterial isolates that are resistant to at least one agent in at least three classes of antibiotics are classified as multidrug-resistant (MDR), while isolates that are responsive to just one or two antimicrobial classes are classified as extensively drug-resistant (XDR) [21]. Drug-resistant A. baumannii infections are significantly harder to treat due to the development of drug resistance brought on by its distinct physiological traits. Multiple antibiotic resistance index (MAR) was determined using the formula “MAR = x/y, where x was the number of antibiotics to which test isolate displayed resistance and y was the total number of antibiotics to which the test organism has been tested” [28].

Previous studies from other hospitals in Egypt confirmed a high prevalence of MDR A. baumannii isolates. It was 100%, 100%, and 61.4% in Benha [27], Ain Shams- Cairo [28], and Menoufia [29] University Hospitals respectively. International Medical Center (IMC), Kobry El-Kobba, and Al-Ganzouri Specialized Hospital- Cairo, MDR A. baumannii isolates represented 88.8% of the total A. baumannii isolates [37]. In a Tertiary Care Hospital in Nepal, 91% of A. baumannii isolates were MDR [35]. In Hamad General Hospital, Qatar, 95% of A. baumannii were MDR [36].

The high prevalence rates of MDR A. baumannii isolates worldwide, result from the resistance gene’s ability to transfer and appear anywhere in the hospital setting. A. baumannii has several defense mechanisms that help it avoid being affected by antimicrobial drugs. Among these processes are efflux pumps, which use efflux mechanisms to remove antibiotics from the cell, and changes made to outer-membrane proteins (OMPs) to lessen porin permeability. Furthermore, A. baumannii produces metallo-β-lactamase enzymes, including “Seoul imipenemase (SIM), New Delhi metallo β-lactamase (NDM), Verona integron-mediated metallo-β-lactamase (VIM), and imipenemase (IMP)”, By which antibiotics can be hydrolyzed such as carbapenems, penicillins, cephalosporins, and monobactams [55].

The A. baumannii-resistant strains continue to appear and spread, and there aren’t many effective treatment choices. For this reason, alternative antibiotics have been studied for use in clinical settings. Tetracyclines, such as doxycycline and minocycline, have demonstrated encouraging microbiological and clinical success in the treatment of A. baumannii infections as monotherapy or in combination with other therapies. It has been found that tetracyclines can be successfully used in conjunction with other antibiotics to treat 87.5% of bloodstream infections and 71.9% of respiratory infections [6]. Unfortunately, tetracyclines-resistant A. baumannii are evolving as a result of the efflux pump [7, 9]. The current study demonstrated 71% resistance to tetracycline and 58% to doxycycline.

Previous studies confirmed the spread of tetracyclines-resistant A. baumannii. The frequency of resistance in the studied isolates was 31.7% against tetracycline from three various hospitals in Erbil City -Iraq [56], 43.5%, and 39% against tetracycline and doxycycline respectively in the hospital of Hillah City- Iraq [57], 60%, and 50% against tetracycline and doxycycline respectively in a tertiary care hospital in North India [58], 65.4% against tetracycline in three tertiary hospitals in Jordan [59], 62% against tetracycline in Leiden University Medical Center in Netherland [54], 91.6% against tetracycline in three hospitals in Tehran City-Iran [60], and 96.3% against tetracycline in Mashhad City-Iran [61].

The high prevalence of antibiotic resistance to commonly given antibiotics in the current study worries the healthcare system since it significantly influences patient care and represents one of the main problems in the treatment of life-threatening infections caused by A. baumannii. This may be because these antimicrobial agents are easily accessible, frequently used outside hospitals, and readily available over the counter for self-medication [60].

In our study, the presence of active efflux systems was investigated in tetracycline-resistant A. baumannii isolates. The minimal inhibitory concentration (MIC) of tetracycline was assayed using “the agar dilution method before and after treatment” with the efflux pump inhibitor with carbonylcyanide 3-chlorophenylhydrazone (CCCP). 45% of the isolates showed a reduction of MIC 4-fold or more, and 12.9% showed a 2-fold reduction in the MIC (Table 6; Fig. 5). The difference in MIC results before and after treatment with CCCP showed a significant difference (p < 0.05), as the percentage of tetracycline resistance changed from 67.7% (before CCCP treatment) to 35.5% (after CCCP treatment) (Table 7). The efflux pump genes AdeA, TetB, AdeB, and TetA were detected in 100%, 100%, 95.2%, and 85.7% of tetracycline-resistant A. baumannii isolates (Table 8).

Several previous studies showed similar findings. Mohammed and Arif discovered that adding CCCP reduced the MIC by at least 4-fold in 22.5% of all A. baumannii isolates [49]. Beheshti et al.. also reported that the efflux pump inhibitor reduced the MIC 4-16-fold in the presence of the efflux pump inhibitor in 91.48% of tetracycline-resistant A. baumannii isolates [6]. Ardehali et al.. reported similar results, indicating that CCCP reduced the MIC of 51.25% of tigecycline-resistant A. baumannii isolates by 2-4-fold [10]. Ranjbar et al. reported that CCCP reduced the MIC by 4- to 8-fold for 87%, 90%, and 62% of tetracycline-, doxycycline- and minocycline-resistant A. baumannii isolates [62]. That was consistent with our findings, as active efflux pumps may be implicated in the higher rate of tetracycline resistance in A. baumannii. The significant reduction in MIC levels upon efflux pump inhibition suggests that efflux pump inhibitors could be a viable adjunctive therapy to restore tetracycline efficacy in MDR A. baumannii infections. Future clinical trials are warranted to explore this potential.

Consistent with these findings, a previous study confirmed the prevalence of TetB and TetA genes in at least 50% and 14%-46% of tetracycline-resistant A. baumannii isolates [63]. Another study showed a high prevalence (61.7%) of TetB but not of TetA gene in tetracycline-resistant isolates [6]. Comparably, TetB was found in a notable proportion of A. baumannii isolates (100% and 95%, respectively) in two separate studies conducted in Iran by Meshkat et al. [64] and Mosavat et al. [65]; surprisingly, TetA was not detected in any of the isolates of such studies.

Efflux pump inhibitors such as CCCP are still in preclinical stages and face regulatory approval challenges due to limited data on their safety in humans; future work involves validating these inhibitors in vivo and assessing their compatibility with antibiotics as adjunctive therapies; rigorous testing and demonstration of safety and efficacy are necessary before considering clinical applications [66]. Efflux pump inhibitors like CCCP are known to disrupt proton gradients, which raises concerns about their toxicity and potential off-target effects. Although it effectively lowers the MIC of some antibiotics, its application is limited due to its nonspecific mechanism and potential for cellular toxicity, studies highlight the need for alternative, safer efflux pump inhibitors with similar efficacy but better safety profiles [67]. Recent studies have shown that CCCP successfully lowers antibiotic resistance in A. baumannii by considerably reducing the MIC of drugs such as cefepime and imipenem. This emphasizes its potential for use in combination therapy to prevent multidrug resistance. However, more research is needed to better understand its specific use and mechanisms of action [11].

Our study recommends strict adherence to infection control procedures to stop the spread and emergence of MDR Acinetobacter spp. isolates, particularly in hospitals. Tetracyclines should be used with active efflux pump inhibitors to treat MDR Acinetobacter spp. infections. We also advise hospitals to use tetracycline medications judiciously and implement antibiotic stewardship initiatives. We will expand our study in further research to encompass resistance patterns in a variety of patient demographics, including age, gender, and underlying diseases; specimen types, such as blood, sputum, and urine; and hospital wards, such as intensive care unit (ICU) versus non-ICU. This strategy will evaluate the effects of clinical and demographic variables on efflux pump expression and resistance mechanisms using a larger, multicenter sample pool. To provide a more thorough knowledge of multidrug resistance in A. baumannii, we will also investigate how efflux pumps interact with other resistance mechanisms, such as enzymatic degradation and porin changes. Our findings will become more clinically relevant and applicable as a result of these efforts.

Our study still has some limitations, (1) the small sample size of the study; (2) including the single-center design where samples were collected from Suez Canal University Hospitals in Ismailia, Egypt only; (3): the focus on a specific subset of resistance mechanisms. Further broader, multicenter studies are needed to identify other types of efflux pumps and other resistance mechanisms and assess their distribution and role in tetracycline-resistant A. baumannii isolates emergence and spread.

Conclusion

This study successfully identified the distribution of AdeAB, TetA, and TetB genes among MDR A. baumannii isolates from Suez Canal University Hospitals, highlighting the significant role of efflux pumps in mediating tetracycline resistance. We also found that CCCP, as an efflux pump inhibitor, can reduce tetracycline’s MIC effectively suggesting that efflux pump inhibitors could be a viable adjunctive therapy to restore tetracycline efficacy.

Data availability

All relevant data are included in this published article.

References

  1. Ahuatzin-Flores OE, Torres E, Chávez-Bravo E. Acinetobacter baumannii, a Multidrug-Resistant Opportunistic Pathogen ihabitatsbiaasystematicereviewReview. Microorganisms. 2024;12(4):644.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  2. Coyne S, Guigon G, Courvalin P, Périchon B. Screening and quantification of the expression of antibiotic resistance genes in Acinetobacter baumannii with a microarray. Antimicrob Agents Chemother. 2010;54(1):333–40.

    Article  CAS  PubMed  Google Scholar 

  3. Stanley CN, Awanye AM, Ogbonnaya UC. Acinetobacter baumannii: Epidemiology, Clinical Manifestations and Associated Infections. In Acinetobacter baumannii-The Rise of a Resistant Pathogen 2023 Nov 10. IntechOpen.

  4. Reina R, León-Moya C, Garnacho-Montero J. Treatment of Acinetobacter baumannii severe infections. Med Intensiva (English Edition). 2022;46(12):700–10.

    Article  CAS  Google Scholar 

  5. Aladel RH, Abdalsameea SA, Badwy HM, Refat SA, ElKholy RM. Role of AdeB gene in multidrug-resistance Acinetobacter. Menoufia Med J. 2020;33(1):205.

    Article  Google Scholar 

  6. Beheshti M, Ardebili A, Beheshti F, Lari AR, Siyadatpanah A, Pournajaf A, Gautam D, Dolma KG, Nissapatorn V. Tetracycline resistance mediated by tet efflux pumps in clinical isolates of Acinetobacter baumannii. Volume 62. Revista do Instituto de Medicina Tropical de São Paulo; 2020. p. 88.

  7. Abdi SN, Ghotaslou R, Ganbarov K, Mobed A, Tanomand A, Yousefi M, Asgharzadeh M, Kafil HS. Acinetobacter baumannii efflux pumps and antibiotic resistance. Infection and drug resistance. 2020 Feb 12:423–34.

  8. Wieczorek P, Sacha P, Hauschild T, Zórawski M, Krawczyk M, Tryniszewska E. Multidrug resistant Acinetobacter baumannii–the role of AdeABC (RND family) efflux pump in resistance to antibiotics. Folia Histochem Cytobiol. 2008;46(3):257–67.

    Article  CAS  PubMed  Google Scholar 

  9. Foong WE, Wilhelm J, Tam HK, Pos KM. Tigecycline efflux in Acinetobacter baumannii is mediated by TetA in synergy with RND-type efflux transporters. J Antimicrob Chemother. 2020;75(5):1135–9.

    Article  CAS  PubMed  Google Scholar 

  10. Zack KM, Sorenson T, Joshi SG. Types and mechanisms of Efflux Pump Systems and the potential of Efflux Pump Inhibitors in the restoration of Antimicrobial susceptibility, with a special reference to Acinetobacter baumannii. Pathogens. 2024;13(3):197.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Sanchez-Carbonel A, Mondragón B, López-Chegne N, Peña-Tuesta I, Huayan-Dávila G, Blitchtein D, Carrillo-Ng H, Silva-Caso W, Aguilar-Luis MA, del Valle-Mendoza J. The effect of the efflux pump inhibitor Carbonyl Cyanide m-Chlorophenylhydrazone (CCCP) on the susceptibility to imipenem and cefepime in clinical strains of Acinetobacter baumannii. PLoS ONE. 2021;16(12):e0259915.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Clinical. and Laboratory Standards Institute (CLSI), Available at: https://clsi.org/.

  13. World Health Organization. Guidelines on standard operating procedures for microbiology. WHO Regional Office for South-East Asia. 2000, Available at: https://iris.who.int/handle/10665/205200

  14. Siegel JD, Rhinehart E, Jackson M, Chiarello L, and the Healthcare Infection Control Practices Advisory Committee., 2007 Guideline for Isolation Precautions: Preventing Transmission of Infectious Agents in Healthcare Settings. Available at: https://www.cdc.gov/infection-control/hcp/isolation/isolation-precautions/index.html

  15. Brown S, Young HK, Amyes SG. Characterization of OXA-51, a novel class D carbapenemase found in genetically unrelated clinical strains of Acinetobacter baumannii from Argentina. Clin Microbiol Infect. 2005;11(1):15–23.

    Article  CAS  PubMed  Google Scholar 

  16. Tawfeeq HR, Rasheed MN, Hassan RH, Musleh MH, Nader MI. Molecular detection of blaoxa genes in Acinetobacter baumannii collected from patients with various infections. Biochem Cell Arch. 2020;20(1):1233–9.

    Google Scholar 

  17. Clinical and Laboratory Standards Institute (CLSI). Performance standards for Antimicrobial susceptibility testing: informational supplement M 100. 33rd ed. USA: Wayne, PA; 2023.

    Google Scholar 

  18. Sun JR, Perng CL, Lin JC, Yang YS, Chan MC, Chang TY, Lin FM, Chiueh TS. AdeRS combination codes differentiate the response to efflux pump inhibitors in tigecycline-resistant isolates of extensively drug-resistant Acinetobacter baumannii. Eur J Clin Microbiol Infect Dis. 2014;33:2141–7.

    Article  CAS  PubMed  Google Scholar 

  19. Roca I, Marti S, Espinal P, Martínez P, Gibert I, Vila J. CraA, a major facilitator superfamily efflux pump associated with chloramphenicol resistance in Acinetobacter baumannii. Antimicrob Agents Chemother. 2009;53(9):4013–4.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Migliaccio A, Esposito EP, Bagattini M, Berisio R, Triassi M, De Gregorio E, Zarrilli R. Inhibition of AdeB, AceI, and AmvA efflux pumps restores chlorhexidine and benzalkonium susceptibility in Acinetobacter baumannii ATCC 19606. Front Microbiol. 2022;12:790263.

    Article  PubMed Central  PubMed  Google Scholar 

  21. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-Liljequist B, Paterson DL. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012;18(3):268–81.

    Article  CAS  PubMed  Google Scholar 

  22. Cockerill FR, Wikler M, Bush K, Dudley M, Eliopoulos G, Hardy D. Clinical and laboratory standards institute. Performance standards for antimicrobial susceptibility testing: twenty-second informational supplement. 2012.

  23. Pumbwe L, Glass D, Wexler HM. Efflux pump overexpression in multiple-antibiotic-resistant mutants of Bacteroides fragilis. Antimicrob Agents Chemother. 2006;50(9):3150–3.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  24. Lannan FM, O’conor DK, Broderick JC, Tate JF, Scoggin JT, Moran NA, Husson CM, Hegeman EM, Ogrydziak CE, Singh SA, Vafides AG. Evaluation of virulence gene expression patterns in Acinetobacter baumannii using quantitative real-time polymerase chain reaction array. Mil Med. 2016;181(9):1108–13.

    Article  PubMed  Google Scholar 

  25. Su HC, Ying GG, Tao R, Zhang RQ, Fogarty LR, Kolpin DW. Occurrence of antibiotic resistance and characterization of resistance genes and integrons in Enterobacteriaceae isolated from integrated fish farms in south China. J Environ Monit. 2011;13(11):3229–36.

    Article  PubMed  Google Scholar 

  26. Baumann P, Doudoroff M, Stanier R. A study of the Moraxella group II. Oxidative-negative species (genus Acinetobacter). J Bacteriol. 1968;95(5):1520–41.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. ALTabbakh AM, Saied SA, Shahat A. Detection of genes of Efflux Pumps (adeB, adeJ and adeG) in Tigecycline Resistant Acinetobacter baumannii isolated from Benha University Hospital. Egypt J Med Microbiol. 2022;31(4):131–9.

    Article  Google Scholar 

  28. Tolba S, El Shatoury EH, Abo AlNasr NM. Prevalence of carbapenem resistant Acinetobacter baumannii (CRAB) in some Egyptian hospitals: evaluation of the use of blaOXA-51-like gene as species specific marker for CRAB. Egypt J Bot. 2019;59(3):723–33.

    Google Scholar 

  29. Badwy HM, Aladel RH, ElKholy RM, Refat SA, Abdalsameea SA. Role of AdeB gene in multidrug-resistance Acinetobacter. Menoufia Med J. 2020;33(1):205–9.

    Article  Google Scholar 

  30. Fouad M, Attia SA, Tawakkol MW, Hashem MA. Emergence ofcarbapenem–resistant Acinetobacter baumannii harboring the OXA–23 gene inintensive care units of Egyptian hospitals. Int J Inf Dis. 2013;17:1775.

    Article  Google Scholar 

  31. Baker AM, Makled AF, Salem EH, Salama AA, Ajlan SE. Phenotypic and molecular characterization of clinical Acinetobacter isolates from Menoufia University Hospitals. Menoufia Med J. 2017;30:1030.

    Article  Google Scholar 

  32. Uwingabiye J, Mohammed F, Abdelhay L, Adil M. Acinetobacter infections prevalenc and frequency of the antibiotics resistance: comparative study of intensive care units vs other hospital units. Pan Afr Med J. 2016;10:116–204.

    Google Scholar 

  33. 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. J Pathogens. 2018;10:1155.

    Google Scholar 

  34. Shrestha S, Tada T, Shrestha B, Ohara H, Kirikae T, Rijal BP, Pokhrel BM, Sherchand JB. Phenotypic characterization of multidrug-resistant Acinetobacter baumannii with special reference to metallo-β-lactamase production from hospitalized patients in a tertiary care hospital in Nepal. J Inst Med. 2015;37(3).

  35. Yadav SK, Bhujel R, Hamal P, Mishra SK, Sharma S, Sherchand JB. Burden of multidrug-resistant Acinetobacter baumannii infection in hospitalized patients in a tertiary care hospital of Nepal. Infection and drug resistance. 2020 Mar 3:725–32.

  36. Al Samawi MS, Khan FY, Eldeeb Y, Almaslamani M, Alkhal A, Alsoub H, Ghadban W, Howady F, Hashim S. Acinetobacter infections among adult patients in Qatar: a 2-year hospital-based study. Canadian Journal of Infectious Diseases and Medical Microbiology. 2016;2016.

  37. Merkier AK, Centrón D. blaOXA-51-type β-lactamase genes are ubiquitous and vary within a strain in Acinetobacter baumannii. Int J Antimicrob Agents. 2006;28(2):110–3.

    Article  CAS  PubMed  Google Scholar 

  38. Daef EA, Mohamed IS, Ahmed AS, El-Gendy SG, Sayed IM. Detection of outbreak caused by multi-drug resistant Acinetobacter baumannii in Assiut University Hospitals. Afr J Microbiol Res. 2014;8(22):2238–44.

    Article  CAS  Google Scholar 

  39. Hassan RM, Salem ST, Hassan SI, Hegab AS, Elkholy YS. Molecular characterization of carbapenem-resistant Acinetobacter baumannii clinical isolates from Egyptian patients. PLoS ONE. 2021;16(6):e0251508.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  40. Bakour S, Alsharapy SA, Touati A, Rolain JM. Characterization of Acinetobacter baumannii clinical isolates carrying blaOXA-23 carbapenemase and 16S rRNA methylase armA genes in Yemen. Microb Drug Resist. 2014;20:604–9.

    Article  CAS  PubMed  Google Scholar 

  41. Vali L, Dashti K, Opazo-Capurro AF, Dashti AA, Al Obaid K, Evans BA. Diversity of multi-drug resistant Acinetobacter baumannii population in a major hospital in Kuwait. Front Microbiol. 2015;6:1–8.

    Article  Google Scholar 

  42. Zowawi HM, Sartor AL, Sidjabat HE, Balkhy HH, Walsh TR, Al Johani SM, AlJindan RY, Alfaresi M, Ibrahim E, Al-Jardani A, Al Salman J. Molecular epidemiology of carbapenem-resistant Acinetobacter baumannii isolates in the Gulf Cooperation Council States: dominance of OXA-23-type producers. J Clin Microbiol. 2015;53(3):896–903.

    Article  PubMed Central  PubMed  Google Scholar 

  43. Rolain J, Loucif L, Elmagboul E, Shaukat A, Ahmedullah H. Emergence of multidrug-resistant Acinetobacter baumannii producing OXA-23 carbapenemase in Qatar. New Microbes New Infect. 2016;1:47–51.

    Article  Google Scholar 

  44. Evans BA, Amyes SG. OXA β-lactamases. Clin Microbiol Rev. 2014;27(2):241–63.

    Article  PubMed Central  PubMed  Google Scholar 

  45. Higgins P, Dammhayn C, Hackel M, Seifert H. Global spread of carbapenem-resistant Acinetobacter baumannii. J Antimicrob Chemother. 2010;65:233–8. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/jac/dkp428.

    Article  CAS  PubMed  Google Scholar 

  46. Kafshnouchi M, Safari M, Khodavirdipour A, Bahador A, Hashemi SH, Alikhani MS, Saidijam M, Alikhani MY. Molecular detection of blaOXA-type carbapenemase genes and antimicrobial resistance patterns among clinical isolates of Acinetobacter baumannii. Global Med Genet. 2022;9(02):118–23.

    Article  Google Scholar 

  47. Ramadan AE, Elgazar AS, Amin NA, Allam AH, Elmahdy MA, Eldeen NA, Saied SA, Shaker DA. Efflux pumps encoding genes (adeA and adeS) in relation to antibiotic resistance pattern in Acinetobacter baumannii strains isolated from Benha university hospital. Egypt J Chest Dis Tuberculosis. 2024;73(3):241–7.

    Article  Google Scholar 

  48. Sanchez-Urtaza S, Ocampo-Sosa A, Molins-Bengoetxea A, El-Kholy MA, Hernandez M, Abad D, Shawky SM, Alkorta I, Gallego L. Molecular characterization of multidrug resistant Acinetobacter baumannii clinical isolates from Alexandria, Egypt. Front Cell Infect Microbiol. 2023;13:1208046.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  49. Mohammed NH, Arif SK. Study the role of the Efflux Pump in Multidrug-Resistant Acinetobacter baumannii. Tikrit J Pure Sci. 2023;28(2):1–1.

    Article  Google Scholar 

  50. Meshkat Z, Salimizand H, Amini Y, Mansury D, Zomorodi AR, Avestan Z, Jamee A, Falahi J, Farsiani H, Mojahed A. Detection of efflux pump genes in multiresistant Acinetobacter baumannii ST2 in Iran. Acta Microbiol Immunol Hung. 2021;68(2):113–20.

    Article  CAS  PubMed  Google Scholar 

  51. Rafiei E, Shahini Shams Abadi M, Zamanzad B, Gholipour A. The frequency of efflux pump genes expression in Acinetobacter baumannii isolates from pulmonary secretions. AMB Express. 2022;12(1):103.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  52. Gharaibeh MH, Abandeh YM, Elnasser ZA, Lafi SQ, Obeidat HM, Khanfar MA. Multi-drug resistant Acinetobacter baumannii: phenotypic and genotypic resistance profiles and the associated risk factors in teaching hospital in Jordan. J Infect Public Health. 2024;17(4):543–50.

    Article  PubMed  Google Scholar 

  53. Kor SB, Tou B, Chieng C, Hiew M, Chew CH. Distribution of the multidrug efflux pump genes adeA, adeI, adeJ, adeY and integrons in clinical isolates of Acinetobacter baumannii from Malaysian hospitals. Biomed Res. 2014;25(2):1–6.

    Google Scholar 

  54. Nemec A, Maixnerová M, van der Reijden TJ, Van den Broek PJ, Dijkshoorn L. Relationship between the AdeABC efflux system gene content, netilmicin susceptibility and multidrug resistance in a genotypically diverse collection of Acinetobacter baumannii strains. J Antimicrob Chemother. 2007;60(3):483–9.

    Article  CAS  PubMed  Google Scholar 

  55. Elbehiry A, Marzouk E, Moussa I, Mushayt Y, Algarni AA, Alrashed OA, Alghamdi KS, Almutairi NA, Anagreyyah SA, Alzahrani A, Almuzaini AM. The prevalence of multidrug-resistant Acinetobacter baumannii and its vaccination status among healthcare providers. Vaccines. 2023;11(7):1171.

    Article  PubMed Central  PubMed  Google Scholar 

  56. Sehree MM, Abdullah HN, Jasim AM. Isolation and evaluation of clinically important Acinetobacter baumannii from intensive care unit samples. J Techniques. 2021;3(3):83–90.

    Article  Google Scholar 

  57. Al-ammery RK, Chabuck ZA. Naturally derived efflux pump inhibitor among tetracycline-resistant Acinetobacter baumannii isolates. Med J Babylon. 2024;21(1):57–64.

    Article  Google Scholar 

  58. Mahich S, Angurana SK, Suthar R, Sundaram V, Munda VS, Gautam V. Acinetobacter sepsis among out-born neonates admitted to neonatal unit in pediatric emergency of a tertiary care hospital in North India. Indian J Pediatr. 2021;88:127–33.

    Article  PubMed  Google Scholar 

  59. Al-Tamimi M, Albalawi H, Alkhawaldeh M, Alazzam A, Ramadan H, Altalalwah M, Alma’aitah A, Al Balawi DA, Shalabi S, Abu-Raideh J, Khasawneh AI. Multidrug-resistant Acinetobacter baumannii in Jordan. Microorganisms. 2022;10(5):849.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  60. Basatian-Tashkan B, Niakan M, Khaledi M, Afkhami H, Sameni F, Bakhti S, Mirnejad R. Antibiotic resistance assessment of Acinetobacter baumannii isolates from Tehran hospitals due to the presence of efflux pumps encoding genes (adeA and adeS genes) by molecular method. BMC Res Notes. 2020;13:1–6.

    Article  Google Scholar 

  61. Sarhaddi N, Soleimanpour S, Farsiani H, Mosavat A, Dolatabadi S, Salimizand H, Jamehdar SA. Elevated prevalence of multidrug-resistant Acinetobacter baumannii with extensive genetic diversity in the largest burn centre of northeast Iran. J Global Antimicrob Resist. 2017;8:60–6.

    Article  Google Scholar 

  62. Ranjbar R, Farahani A. Study of genetic diversity, biofilm formation, and detection of Carbapenemase, MBL, ESBL, and tetracycline resistance genes in multidrug-resistant Acinetobacter baumannii isolated from burn wound infections in Iran. Antimicrob Resist Infect Control. 2019;8:1–1.

    Article  Google Scholar 

  63. Coyne S, Courvalin P, Périchon B. Efflux-mediated antibiotic resistance in Acinetobacter Spp. Antimicrob Agents Chemother. 2011;55(3):947–53.

    Article  CAS  PubMed  Google Scholar 

  64. Meshkat Z, Salimizand H, Amini Y, Khakshoor M, Mansouri D, Farsiani H, Najafi A. Molecular characterization and genetic relatedness of clinically Acinetobacter Baumanii isolates conferring increased resistance to the first and second generations of tetracyclines in Iran. Ann Clin Microbiol Antimicrob. 2017;16:51.

    Article  PubMed Central  PubMed  Google Scholar 

  65. Mosavat A, Soleimanpour S, Farsiani H, Salimizand H, Kebriaei A, Jamehdar SA, Bagheri M, Rezaee SA, Ghazvini K. Moderate genetic diversity with extensive antimicrobial resistance among multidrug-resistant Acinetobacter baumannii in a referral hospital in Northeast Iran. Jundishapur Journal of Microbiology. 2018;11(7).

  66. Zhang L, Tian X, Sun L, Mi K, Wang R, Gong F, Huang L. Bacterial efflux pump inhibitors reduce antibiotic resistance. Pharmaceutics. 2024;16(2):170.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  67. Verma P, Tiwari M, Tiwari V. Efflux pumps in multidrug-resistant Acinetobacter baumannii: current status and challenges in the discovery of efflux pumps inhibitors. Microb Pathog. 2021;152:104766.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Non.

Funding

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

None.

Author information

Authors and Affiliations

  1. Microbiology and Immunology Department, Faculty of Medicine, Suez Canal University, Ismailia, Egypt

    Hasnaa Azab & Sally Khattab

  2. Clinical and Chemical Pathology Department, Faculty of Medicine, Suez Canal University, Ismailia, Egypt

    Aya Mohamed Askar

  3. Medical Biochemistry and Molecular Biology Department, Faculty of Medicine, Suez Canal University, Ismailia, Egypt

    Noha M. Abd El-Fadeal

  4. Biochemistry Department, Ibn Sina National College for Medical Studies, Kingdom of Saudi Arabia, Jeddah, 22421, Saudi Arabia

    Noha M. Abd El-Fadeal

  5. Oncology Diagnostic Unit, Faculty of Medicine, Suez Canal University, Ismailia, Egypt

    Noha M. Abd El-Fadeal

  6. Internal Medicine Department, Faculty of Medicine, Suez University, Suez, 43511, Egypt

    Amira A. A. Othman

  7. Department of Basic Medical Science, College of Medicine, AlMaarefa University, Riyadh, Saudi Arabia

    Amal H. Rayan

  8. Faculty of Medicine, Suez Canal University, Ismailia, Egypt

    Amal H. Rayan

Authors
  1. Hasnaa Azab
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Aya Mohamed Askar
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Noha M. Abd El-Fadeal
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Amira A. A. Othman
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Amal H. Rayan
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Sally Khattab
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

H. Azab: concept and design of the study, data acquisition, statistical analysis, interpreted the results, analyzed the data, drafted the manuscript, critically revised the manuscript, approved the final version to be published, and agreed to be accountable for all aspects of the work. A. M. Askar: concept and design of the study, data acquisition, analyzed the data, drafted the manuscript, critically revised the manuscript, and approved the final version to be published. N. M. Abd El-Fadeal: concept and design of the study, data acquisition, analyzed the data, drafted the manuscript, critically revised the manuscript, and approved the final version to be published. A.A.A. Othman: concept and design of the study, interpreted the results, analyzed the data, drafted the manuscript, critically revised the manuscript, and approved the final version to be published. A. H. Rayan: concept and design of the study, interpreted the results, analyzed the data, drafted the manuscript, critically revised the manuscript, and approved the final version to be published. S. Khattab: concept and design of the study, interpreted the results, analyzed the data, drafted the manuscript, critically revised the manuscript, and approved the final version to be published.

Corresponding author

Correspondence to Amira A. A. Othman.

Ethics declarations

Human ethics and consent to participate

Ethical approval was obtained from the Research Ethics Committee of the Faculty of Medicine, Suez Canal University, Egypt, #5024. All subjects were informed and gave their voluntary, written informed consent. It is declared that: All methods were carried out by relevant guidelines and regulations. It was performed according to the recommendations of Good Clinical Practice and the Declaration of Helsinki (2013).

Consent for publication

N/A.

Competing interests

The authors declare no competing interests.

Clinical trials registry

Pan African Clinical Trials Registry.

Trial No.: PACTR202410825114244.

Trial URL: https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=31981.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Azab, H., Askar, A.M., El-Fadeal, N.M.A. et al. Detection of AdeAB, TetA, and TetB efflux pump genes in clinical isolates of tetracycline-resistant Acinetobacter baumannii from patients of Suez Canal University Hospitals. BMC Microbiol 25, 63 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12866-024-03735-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12866-024-03735-1

Keywords

BMC Microbiology

ISSN: 1471-2180

Contact us