Summary
Introduction: The increasing emergence of multidrug-resistant (MDR) Acinetobacter infections has become a significant challenge for physicians and clinical microbiologists owing to the difficulties arising during therapy. The major efflux mechanism associated with MDR in A. baumannii is the chromosomally encoded tripartite efflux pump, AdeABC, which has been reported worldwide. AdeABC belongs to the resistance-nodulation-division efflux pump family and has a three-component structure: AdeB forms the transmembrane component, AdeA forms the inner membrane fusion protein, and AdeC forms the outer membrane protein. AdeABC is chromosomally encoded and is regulated by a two-component system containing a sensor kinase (AdeS) and its associated response regulator (AdeR). Point mutations in these components are associated with the overexpression of AdeABC, thereby leading to multiple drug resistance. The purpose of this study was to investigate the distribution of the AdeABC efflux pump genes and their relationship with carbapenem and multiple drug resistance in A. baumannii strains isolated from the blood cultures of hospitalized patients.
Materials and Methods: A total of 97 A. baumannii strains that were isolated from the blood cultures of hospitalized patients in different departments, were included in the study. The Phoenix Automated System was used to identify and determine antibiotic susceptibility patterns. The susceptibility of the study strains to carbapenems, ciprofloxacin, trimethoprim-sulfamethoxazole, amikacin, gentamicin, and netilmicin were determined according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. AdeRS mutations and adeB gene expression of drug efflux genes were analyzed by sequencing and qPCR, respectively. The 16S rRNA gene was used as a housekeeping gene, and the A. baumannii ATCC 19606 standard strain was also used to normalize the expression results of adeB gene.
Results: Of the 97 isolates, 61 were found to be carbapenem resistant. The resistance rates of carbapenem-resistant A. baumannii (CRAB) isolates were found to be 100% for ceftazidime; 96.7% for cefepime, piperacillin-azobactam, ciprofloxacin, and trimethoprim-sulfamethoxazole; 86.8% for amikacin; and 75.4% for gentamicin and netilmicin. The significant overexpression (3.45-52.18 fold) of adeB was observed in 49 CRAB isolates, whereas less increased levels were observed in only 12 CRAB isolates (0.23-0.54 fold) and non-CRAB isolates (0.109-0.783 fold). In total, 80.3% of the CRAB isolates were positive for the adeRS genes. The p.Val120Ile change in the AdeR aminoacid sequence was determined in 42.8% of the adeB-overexpressing CRAB isolates. The p.His158Leu and p.Pro116Ser changes were found in 36.7% of these isolates. None of the non-CRAB isolates had p.Val120Ile, p.His158Leu, and p.Pro116Ser changes. In the AdeS aminoacid sequence, p.Gly293Ser, p.Leu105Phe, and His227Asp changes were most commonly observed in adeB-overexpressing CRAB isolates, whereas pGly293Ser change was detected in only 8% of the non-CRAB isolates.
Conclusion: These data showed that AdeABC efflux pump overexpression (both adeB expression and AdeRS mutation) was higher than expected in our A. baumannii isolates. They were significantly associated with the AdeABC efflux system and both CRAB and MDR isolates. The overexpression of adeB and aminoacid changes in the AdeRS regions led to an increase resistance to different antibiotics; therefore, A. baumannii strains should be monitored to ensure the correct treatment, especially in nosocomial MDR.
Introduction
Acinetobacter baumannii is a ubiquitous, Gram-negative coccobacillus, which is an important nosocomial pathogen that causes various infections, such as wound infections, bloodstream infections, ventilator-acquired pneumonia, central nervous system infections, and urinary tract infections. In fact, A. baumannii is considered as an opportunistic pathogen. The increasing emergence of multidrug-resistant (MDR) Acinetobacter spp. infections has become a significant challenge for physicians and clinical microbiologists due to the difficulties arising during therapy[1-4]. The major efflux mechanism associated with MDR in A. baumannii is the chromosomally encoded tripartite efflux pump, AdeABC, which has been reported globally. AdeABC belongs to the resistance-nodulation-division efflux pump family and has a three-component structure: AdeB forms the transmembrane component, AdeA forms the inner membrane fusion protein, and AdeC forms the outer membrane protein. AdeABC is chromosomally encoded and is regulated by a two-component system containing a sensor kinase (AdeS) and its associated response regulator (AdeR). Point mutations in these components are associated with the overexpression of AdeABC, thereby leading to multiple drug resistance. This major efflux mechanism is associated with carbapenems, aminoglycosides, fluoroquinolones, tetracyclines, amphenicols, macrolides, and trimethoprim sulfamethoxazole[5-9].
The purpose of this study was to investigate the distribution of the AdeABC efflux pump genes and their relationship with carbapenems and aminoglycosides susceptibility in A. baumannii strains isolated from the blood cultures of hospitalized patients.
Methods
According to the “Ethical Principles for Medical Research Involving Human Subjects” of the principles of the World Medical Association Declaration of Helsinki (amended in October 2013), the İstanbul University-Cerrahpaşa, Cerrahpaşa Faculty of Medicine Ethics Committee of Clinical Research (decision no: 2015/147) granted the approval to this study. The written informed consent was obtained from the study participants.
Study strains were the 97 A. baumannii strains that were isolated from the blood cultures of hospitalized patients in different departments (intensive care 49%, surgery 19.6%, hematology 9.8%, orthopedics and traumatology 3%, and internal medicine 18%) of İstanbul University-Cerrahpaşa, Cerrahpaşa Faculty of Medicine Hospital, İstanbul, Turkey.
The BD Phoenix™ automated identification and susceptibility pattern-testing system (Becton-Dickinson Company, Franklin Lakes, NJ, USA) was used to identify and determine the antibiotic susceptibility. The concentration gradient-based E-test (bioMérieux, France) strip method was employed to measure the minimum inhibitory concentration (MIC) values in imipenem, meropenem, and colistin in vitro susceptibility tests. The susceptibilities of the strains to carbapenems, ciprofloxacin, trimethoprim-sulfamethoxazole, amikacin, gentamicin, and netilmicin were determined according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria[10].
The ribonucleic acid (RNA) samples were isolated by using the High Pure RNA isolation kit (Roche Diagnostics GmBH, Mannheim, Germany) from A. baumannii strains produced in the Luria Bertani (LB) medium (Sigma-Aldrich, St. Louis, MO, USA) in accordance with the manufacturer’s instructions. The obtained RNA samples were stored at -80 °C until they were processed by qPCR LightCycler 480 II (Roche Diagnostics GmBH). Prior to qPCR runs, the ratios of RNAs at A260/A280 nm were examined on a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) for the calculation of the quantities and purity values. Complementary DNA (cDNA) synthesis was performed by using the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics GmBH) by adhering to the manufacturer’s instructions. Each sample weighed 50 ng. The 16S rRNA gene was used as a housekeeping gene. The primers were ordered from IDT (Integrated DNA Technologies Inc., Skokie, IL, USA) and were used for qPCR steps for the adeB and 16S rRNA genes. These primers are presented in Table 1.
The qPCR experiments were performed by using the LightCycler 480 SYBR Green I Master kit (Roche Diagnostics GmBH) to detect the adeB and 16S rRNA genes in accordance with the manufacturer’s instructions. The qPCR protocol consisted of enzyme activation for 10 min at 95 °C. After 45 cycles, there was an amplification phase of 10 s at 95 °C, 20 s at 60 °C, and 3 s at 72 °C, which was followed by 1 s of denaturation at 95 °C, 60 s at 65 °C, and continuous reading up to 97 °C. The fluorescence data were obtained automatically, and the adeB and 16S rRNA Cp values were generated for each isolate by using the ΔCt method. The A. baumannii ATCC 19606 standard strain was also used to normalize the adeB gene expression results[11]. Each isolate was tested in duplicate samples in two independent experiments.
Deoxyribonucleic acid (DNA) isolations were performed by using the High Pure PCR Template preparation kit (Roche Diagnostics GmBH) from the A. baumannii strains generated in the LB medium in accordance with the manufacturer’s instructions. The obtained DNA sample was stored at -20 °C until the process of sequencing. DNA sequencing for the detection of mutations in the adeR and adeS genes was performed by using the primers shown in Table 1. Primers were obtained from IDT (Integrated DNA Technologies Inc.). DNA sequencing was performed by using the automated MegaBACE 1000 (Amersham Biosciences, CA, USA) sequencing system in accordance with the manufacturer’s instructions. At the end of the process, the chromatogram files obtained for the AdeS and AdeR gene regions were compared with the sequences obtained by downloading them after being converted to the FASTA format (See Supplementary Table 1 for GenBank Accession Numbers-placed after the references).
Results
Of the 97 isolates, 61 were found to be carbapenem resistant. The MIC values were found to range between 8-12 mg/ml for imipenem and meropenem.
The resistance rates of carbapenem-resistant A. baumannii (CRAB) isolates were found to be 100% for ciprofloxacin and trimethoprim-sulfamethoxazole, 86.8% for amikacin, and 75.4% for gentamicin and netilmicin (Figure 1). The significant overexpression (3.45-52.18 fold) of adeB was observed in the 49 CRAB isolates, whereas only 12 CRAB isolates (0.23-0.54 fold) and non-CRAB isolates (0.109-0.783 fold) had less increased levels (Figures 2, 3). Of the 97 isolates, 42 were resistant to aminoglycosides. The significant overexpression (21.38-51.68 fold) of adeB was displayed in the 34 aminoglycoside-resistant isolates and was found to be positive for the AdeRS gene. Eight aminoglycoside-resistant isolates had low levels (0.23-0.54) of adeB, which were found to be negative for the adeRS gene (Table 2).
Approximately 80.3% of the CRAB isolates were found to be positive for the adeRS gene, the p.Val120Ile change in the AdeR amino acid sequence was determined in the 21 (42.8%) isolates of adeB-overexpressing CRAB isolates. In total, 14 of these isolates were aminoglycoside-resistant isolates. The p.His158Leu and p.Pro116Ser changes were observed in 36.7% of the CRAB isolates. Eight of the aminoglycoside-resistant isolates showed change in p.His158Leu, and 15 of them showed p.Pro116Ser change. None of the non-CRAB isolates and aminoglycoside-susceptible isolates showed p.Val120Ile, p.His158Leu, and p.Pro116Ser changes. In the AdeS amino acid sequence, p.Gly293Ser, p.Leu105Phe, and p.His227Asp changes were most commonly observed in the adeB-overexpressing CRAB isolates and aminoglycoside-resistant isolates. The p.Gly293Ser change was detected in only 8% of the non-CRAB isolates (Table 3).
Discussion
The RND family is a multidrug efflux pump and plays a vital role in the antimicrobial resistance of A. baumannii. The first characterized RND system in A. baumannii samples was the AdeABC efflux pump. The expression is controlled by the two-component regulatory system known as adeS and adeR[12-14]. The MDR phenotype against antimicrobials is more expressed than the natural isolates at this pump[13, 15].
Aminoglycosides are the antibiotics most affected by the ABC-type pumps[16-18]. For this reason, we tried to measure the expression level of adeB gene and investigate the mutation of adeR and adeS genes, the regulatory compartments, and their relationship with carbapenemase production in A. baumannii samples isolated from the blood cultures of hospitalized patients. This study found that the adeB gene expression increased 52-fold with p.Val120Ile aminoacid change. In addition, there was a 50-fold increase with p.Pro116Ser and p.His158Leu aminoacid changes for the AdeR region. The aminoacid changes of p.Gly293Ser, p.Leu105Phe, and p.His227Asp were observed most frequently in the cases of adeB overexpression for the AdeS region. The most frequent p.Val120Ile change was observed in the 97 isolates for the AdeR region, whereas the most frequent p.Gly293Ser change was observed for the AdeS region.
Qiu et al.[19] reported that the adeB expression of CRAB isolates was 10.4-62.3 times higher than that of non-CRAB isolates. Our study found similar results. The CRAB isolates had at least 3.45-52.18 times higher overexpression pattern for the adeB gene. Coyne et al.[20] reported that the adeB overexpressing strains were less susceptible to gentamicin and had a 12-fold increase in MICs. We found similar resistance results for amikacin, gentamicin, and netilmicin, and the adeB overexpression in this resistant isolates was found to be 21.38-51.68 fold higher than that in the susceptible isolates. Lari et al.[21] suggested that the efflux-based system AdeABC was an important contributor to reduced susceptibility to antibiotics of choice for treatment, including ciprofloxacin and cefepime, in the A. baumannii isolates.
Ardebili et al.[22] reported that p.His158Leu, p.Pro116Ser, p.Val120Ile, and p.Ala136Val were the most common aminoacid changes in the AdeR regions, whereas the p.Lys84Glu, p.Ala97Ser, and p.Gly103Asp were the most common aminoacid changes in the AdeS regions. They also reported that these changes had caused increased ciprofloxacin MICs, similar with aminoglycosides. We also found similar results for these mutations in ciprofloxacin. Richmond et al.[23] reported that the strains in p.Ala94Val mutation in the AdeS region were observed to have 91-fold higher adeB expression than the non-mutagenic strains. Similarly, several studies have shown the same results[24-26].
To the best of our knowledge, this is the first study in our country to detect both mutations in AdeRS and the expression level in adeB on the clinical A. baumannii isolates.
Conclusion
These results demonstrated that AdeABC efflux pump overexpression (both adeB expression and AdeRS mutation) is higher than expected in our A. baumannii isolates. They were significantly associated with the AdeABC efflux system and both CRAB and MDR isolates. The overexpression of adeB and aminoacid changes in the AdeRS regions lead to an increase in resistance to different antibiotics. Nosocomial A. baumannii strains especially the MDR strains should be monitored to ensure correct treatment.
Ethics
Ethics Committee Approval: The study approved by the Clinical Research Ethics Committee of İstanbul University-Cerrahpaşa, Cerrahpaşa Faculty of Medicine (decision no: 2015/147).
Informed Consent: The written informed consent was obtained from the study participants.
Peer-review: Externally and internally peer-reviewed.
Authorship Contributions
Concept: O.A., H.A., M.D., F.K.Ç., Design: O.A., H.A., M.D., F.K.Ç., Data Collection or Processing: O.A., H.A., M.D., Analysis or Interpretation: M.D., F.K.Ç., Literature Search: O.A., M.D., F.K.Ç., Writing: O.A., M.D., F.K.Ç.
Conflict of Interest: No conflict of interest was declared by the authors.
Financial Disclosure: The authors declared that the study was funded by the Scientific Research Projects Coordination Unit of İstanbul University-Cerrahpaşa (project number: 41923/3547).
