# Efflux Pump Activity and Mutations Driving Multidrug Resistance inAcinetobacter baumannii at a Tertiary Hospital in Pretoria, South Africa

Authors: Noel-David Nogbou; Granny M. Nkawane; Khanyisa Ntshane; Charles K. Wairuri; Dikwata T. Phofa; Kagiso K. Mokgokong; Mbudzeni Ramashia; Maphoshane Nchabeleng; Lawrence C. Obi; Andrew M. Musyoki
Journal: International Journal of Microbiology
 (2021)

---

## Abstract

Acinetobacter baumannii (A. baumannii) has developed several resistance mechanisms. The bacteria have been reported as origin of multiple outbreaks. This study aims to investigate the use of efflux pumps and quinolone resistance-associated genotypic mutations as mechanisms of resistance in A. baumannii isolates at a tertiary hospital. A total number of 103 A. baumannii isolates were investigated after identification and antimicrobial susceptibility testing by VITEK2 followed by PCR amplification of blaOXA-51. Conventional PCR amplification of the AdeABC efflux pump (adeB, adeS, and adeR) and quinolone (parC and gyrA) resistance genes were performed, followed by quantitative real-time PCR of AdeABC efflux pump genes. Phenotypic evaluation of efflux pump expression was performed by determining the difference between the MIC of tigecycline before and after exposure to an efflux pump inhibitor. The Sanger sequencing method was used to sequence the parC and gyrA amplicons. A phylogenetic tree was drawn using MEGA 4.0 to evaluate evolutionary relatedness of the strains. All the collected isolates were blaOXA-51-positive. High resistance to almost all the tested antibiotics was observed. Efflux pump was found in 75% of isolates as a mechanism of resistance. The study detected parC gene mutation in 60% and gyrA gene mutation in 85%, while 37% of isolates had mutations on both genes. A minimal evolutionary distance between the isolates was reported. The use of the AdeABC efflux pump system as an active mechanism of resistance combined with point mutation mainly in gyrA was shown to contribute to broaden the resistance spectrum of A. baumannii isolates.


---

## Body

## 1. Background

Acinetobacter baumannii (A. baumannii) is one of the nosocomial pathogens that exhibits high level of resistance to antibiotics [1, 2]. This Gram-negative coccobacillus bacterium is responsible for infections predominantly in the intensive care unit [3, 4]. The bacterium causes pneumonia, bacteraemia, septicaemia [5], meningitis [6], and urinary tract infections [7] often seen in patients undergoing invasive procedures such as urinary catheter, tracheal intubation, and those with underlying conditions [2]. A. baumannii has demonstrated several antimicrobial resistance mechanisms [8–10] against antibiotics such as aminoglycosides, carbapenems, fluoroquinolones, cephalosporins, tetracyclines, sulbactams, rifampicins [9], as well as colistin [11–13] and tigecycline [8, 14], the so called last resort drugs used to treat its infections [8, 11–14]. Moreover, literature has reported resistance to recently developed antibiotics such as eravacycline [15] and cefiderocol [16]. The bacterium has acquired several resistance mechanisms through mobile genetic elements [17, 18] and has also shown to have natural resistance against some antibiotics including aminopenicillins, first and second generation cephalosporins, aztreonam, ertapenem, fosfomycin, chloramphenicol, and trimethoprim [19] leading to emergence of multidrug and pandrug-resistant strains [8, 9]. The main mechanisms expressed by A. baumannii to escape antibiotics attack are production of antibiotic-hydrolysing enzymes, poor membrane penetration, permeability defects, alteration of target sites or cellular functions, and active efflux pumps [20].Efflux pumps are the main mediators of resistance mechanisms against many antibiotic classes [21]. Through this mechanism, the bacteria avoid accumulation of drugs at the targeted site within it, leading to decreased susceptibility to antibiotics [17, 21]. Three resistance-nodulation-cells division encoded in A. baumannii genome is reported to contribute to antibiotic resistance in A. baumannii clinical isolates [22]. The overexpression of efflux pumps in A. baumannii has been associated with an increased resistance to antibiotics such as tigecycline [14, 23], carbapenems [24], minocycline, gentamycin, doxycycline, and tetracycline [25]. Several researchers support that among the resistance-nodulation-cell division superfamily, the adverse effect of ATP-binding cassette (AdeABC) is the most associated with resistance in A. baumannii [26, 27]. The system generally consists of three parts: multidrug transporter adeB that captures antibiotics in the inner membrane of phospholipids bilayer or cytoplasm, membrane fusion protein adeA that acts as membrane fusion protein, and finally, the outer membrane protein adeC that is a membrane channel protein, used by adeB to transport out the substrates [26]. This whole mechanism is regulated by adeR and adeS, a two-component system [26, 27]. The working mechanism of this system suggests that adeB and the regulatory genes adeR and adeS are the main role players within the AdeABC efflux pump system [28]. This mechanism of resistance reduces the susceptibility of A. baumannii to multiple class of antibiotics [23, 28–30] and has been reported to be associated with resistance to newly developed drugs [30].Alteration of target site or cellular function due to mutations has enabled emergence of resistance to antibiotics within theA. baumannii species [15, 31]. This mechanism of resistance is used by A. baumannii to resist fluoroquinolones [32], colistin [33], novel synthetic beta-lactamase ceftazidime–avibactam [34], and several newly developed antibiotics [35]. Even though literature reports on the plasmid mediated quinolone resistance genes qnrA, qnrB, and qnrS as one of the resistance mechanisms to reduce susceptibility to quinolones [36, 37], A. baumannii are mainly resistant to quinolones through chromosomal gene mutation in parC and gyrA [38, 39] and other molecular mechanisms [37]. Literature supports that a single mutation in gyrA inducing an amino acid change from serine to leucine in position 83 (serine 83) reduces the susceptibility of A. baumannii clinical isolates to fluoroquinolones [40]. Two mutations in parC in position 80 (serine 80) and 84 (Glu-84) inducing change from serine to isoleucine and glutamic acid into valine, respectively, lead to resistance to fluoroquinolones [40].The combined effect of active use of efflux pumps and alteration of target site due to mutations contribute to broaden the resistance spectrum ofA. baumannii strains and pose serious therapeutic challenges to clinicians in establishment of effective treatment regime. Multiple outbreaks of A. baumannii infections have been observed globally with an increased resistance to antimicrobial drugs [41, 42]. In this study, we focused on the use of AdeABC efflux pumps, parC and gyrA mutations, contributing to reduce A. baumannii clinical isolates susceptibility to antibiotics at an academic hospital in Pretoria. The aim was to investigate the use of the efflux pump and quinolone resistance-associated genes in the rise of multidrug resistant A. baumannii strains.


## 2. Materials and Methods

### 2.1. Study Design, Settings, and Samples Collection

Isolates for this study were collected between February 2018 and February 2020 at Dr. George Mukhari Tertiary Laboratory (DGMTL), a unit of the National Health Laboratory Services (NHLS) of South Africa. DGMTL is a level 3 clinical laboratory where routine laboratory diagnostics for patients presenting at Dr. George Mukhari Academic Hospital (DGMAH), and 3 district hospitals and surrounding clinics are performed. DGMTL is coupled with the Department of Microbiological Pathology of Sefako Makgatho Health Sciences University (SMU). Ethical approval to conduct this research was granted by Sefako Makgatho Health Sciences University Research Ethics Committee (SMUREC). A total number of 103A. baumannii isolates were collected from DGMTL and stored at −70oC until use.


### 2.2. Isolate’s Identification and Antimicrobial Susceptibility Profiles

Collected isolates were identified using a phenotypic and genotypic method, VITEK2 automated system (bioMerieux, France) and polymerase chain reaction (PCR) amplification ofblaOXA-51 gene.Antimicrobial susceptibility testing was performed using the VITEK2 automated system (bioMerieux, France). Piperacillin + tazobactam (ptz), ceftazidime (caz), cefepime (fep), trimethoprim/sulfamethoxazole (sxt), gentamycin (cn10), ciprofloxacin (cip), cefotaxime (ctx), imipenem (imp), meropenem (mem), and tigecycline (tig) were tested.


### 2.3. Molecular Investigation of Resistance Mechanisms

#### 2.3.1. Recovery of Isolates and Nucleic Acid Extraction

Recovery of a fresh pure colony on Muller–Hinton (MH) agar (Diagnostic Media Products, DMP, South Africa) was conducted from microbanks (Microbanks™, ProLab Diagnostics Inc., Canada) under aseptic environment. A single pure colony was used for DNA and RNA extraction. DNA was extracted using the boiling method [43]; briefly, a loopful of fresh pure colony was suspended in 1000 µL of saline (SABAX Pour Saline 0.9%, Adcock Ingram Critical Care (Pty) Ltd., South Africa) in an Eppendorf tube (Eppendorf AG, Hamburg, Germany) followed by incubation (Memmert Incubator IN30, Germany) for 20 min at 37°C ± 2. The suspension was then removed and well shaken using a vortex (Vortex, Heidolph, Reax top, Germany) at full speed for 20 s. Thereafter, the suspension was centrifuged (Mikro 20, Werk Nr. Bajahr E Kin, Hettich Zentrifugen, Germany) for 5 min at 130 × 100 rotation per minute (RPM). The supernatant was removed and resuspended in 200 µL of PCR grade water (BioConcept Ltd., Switzerland) in an Eppendorf tube (Eppendorf AG, Hamburg, Germany). The suspension was then put on a thermomixer (Eppendorf, Thermomixer Compact, MERCK, Merck Chemical (Pty) Ltd., South Africa) at 90°C for 10 mn at 700 RPM. Thereafter, it was put on a cell disruptor (Disruptor Genie®, Scientific Industries SI™, USA) for 10 min. Finally, the suspension was centrifuged (Mikro 20, Werk Nr. Bajahr E Kin, Hettich Zentrifugen, Germany) for 10 min at 130 × 100 RPM. The supernatant containing the DNA was transferred and preserved in the sterile Eppendorf tube (Eppendorf AG, Hamburg, Germany) and stored at −20°C.RNA extraction was conducted using a commercial RNA isolation kit (ISOLATE II RNA Mini Kit, MagMAX™ Viral/Pathogen, bioline, London, United Kingdom) following the manufacturer’s instructions [44]. Extracted RNA was quantified using a spectrophotometer (NanoDrop Lite Spectrophotometer, NanoDrop Products, Thermo Scientific, USA) and normalized to 1 μg/μL. The obtained RNA was stored at −80°C. A complementary DNA (cDNA) synthesis was performed using a cDNA synthesis kit (SensiFAST cDNA Synthesis Mix Reaction Guidelines, Bioline, a Meridian Life Science® Company, United Kingdom) following the manufacturer’s instructions. cDNA products were stored at −20°C, until further use.


#### 2.3.2. Conventional PCR Amplification of AdeABC Efflux Pump (adeB, adeS, and adeR) and Quinolone (parC and gyrA) Resistance Genes

Gene amplification by conventional PCR was performed using 2X MyTaq HS Red Mix (MyTaq HS Red Mix, Meridian Bioscience, bioline, United Kingdom) to detect genes of interest inA. baumannii. The Bioline protocol [45] was followed to prepare multiplex PCR (M-PCR) assays using primers with similar melting temperatures and monoplex PCR (m-PCR) for primers of different annealing temperatures. To avoid mistakes during detection of amplicon by gel electrophoresis, the AdeABC efflux pump system associated genes adeB and adeS which differ by only 3 nucleotides in base pairs were ran separately. M-PCR was run for genes adeS and adeR and a m-PCR for gene adeB. PCR was performed in a reaction mixture of a total volume of 25 μL, composed as follows: 12.5 μL of 2X MyTaq HS Red Mix (MyTaq HS Red Mix, Meridian Bioscience, Bioline, United Kingdom), 0.5 μL of each primer, and 6.5 μL PCR grade water (BioConcept Ltd., Switzerland) were added to make up to 20 μL, and 5 μL of DNA template was added to constitute a 25 μL reaction mix. Negative and positive controls were run with every PCR on a thermocycler (GeneAmp® PCR System 2700, Applied Biosystems, Singapore). The thermocycling conditions for conventional PCR and primers used for detection of drug resistance-associated genes are detailed in Supplementary 2.


#### 2.3.3. PCR Amplicon Detection

The detection of PCR amplicons was conducted in a 1% agarose gel stained with ethidium bromide.


#### 2.3.4. Quantitative Real-Time PCR (qRT-PCR) Amplification of AdeABC Efflux Pump (adeB, adeS, and adeR)

The qRT-PCR was conducted on cDNA for the detection of the presence of mRNA, using the SYBR No-ROX kit (SensiFAST™ SYBR® NO-ROX Kit, Meridian Bioscience, Bioline, United Kingdom) on Sacace Real-Time PCR Instrument (SaCycler-96, Real-Time PCR System, Sacace Biotechnologies Srl, Scalabrini, Italy). The qRT-PCR reaction mixture was prepared in a volume of 20 μL comprising of 4 μL of cDNA, 10 μL SYBR, 0.8 μL of each primer, and 4.4 μL of PCR grade water (BioConcept Ltd., Switzerland). The thermocycling conditions and primers used are detailed in Supplementary 1 and 2, respectively.


### 2.4. Phenotypic Evaluation of AdeABC Efflux PumpadeB, adeS, and adeR Gene Expression

A functional AdeABC efflux system was assessed by evaluating the difference between the minimal inhibitory concentrations (MICs) for tigecycline (TGC) using the gradient diffusion method (tigecycline, MIC Test Strip, Liofilchem® Srl, Roseto d’Abruzzi, Italy) before and after exposure to an efflux pump inhibitor (EPI) carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Sigma-Aldrich, Dorset, United Kingdom) [46]. Briefly, CCCP was added to one of two MH agar plates at the final concentration of 100 µg/mL. From a fresh overnight culture of A. baumannii, a 0.5 Mac Farhland turbid sample was made (Densichek, BioMerieux DensiCHEK Plus, USA). A swab was then used to spread A. baumannii on each agar plate, followed by placement of a 0.016–256 mg/L TGC E-strip test in the middle of each agar plate followed by an overnight incubation at 37°C. E. coli strain ATCC 85218 was used as a control.


### 2.5. Sequencing of Quinolone Resistance-Associated Genes (parC and gyrA)

The Sanger sequencing method (ABI3500XL; Applied Biosymptoms, United States) at Inqaba Biotec (Pretoria) was used to sequence theparC and gyrA amplicons. Sequences were edited using ChromasPro software (version 2.0) and then aligned together with wild-type sequences from GenBank using BioEdit (http://www.mbio.ncsu.edu/BioEdit/BioEdit.html). Serine 83 of gyrA gene and serine 80 and serine 84 of parC gene were investigated for mutation. A phylogenetic tree was drawn using molecular evolutionary genetic analysis (MEGA) 4.0 [47] to evaluate evolutionary relatedness of the study strains.


### 2.6. Statistical Analysis

Data were captured on Microsoft Professional Excel 2016 and data analysis conducted on IBM SPSS Statistics 26 (IBM® SPSS® Statistics version 26.0, 2019). Ap value of ≤0.05 was considered statistically significant.


## 2.1. Study Design, Settings, and Samples Collection

Isolates for this study were collected between February 2018 and February 2020 at Dr. George Mukhari Tertiary Laboratory (DGMTL), a unit of the National Health Laboratory Services (NHLS) of South Africa. DGMTL is a level 3 clinical laboratory where routine laboratory diagnostics for patients presenting at Dr. George Mukhari Academic Hospital (DGMAH), and 3 district hospitals and surrounding clinics are performed. DGMTL is coupled with the Department of Microbiological Pathology of Sefako Makgatho Health Sciences University (SMU). Ethical approval to conduct this research was granted by Sefako Makgatho Health Sciences University Research Ethics Committee (SMUREC). A total number of 103A. baumannii isolates were collected from DGMTL and stored at −70oC until use.


## 2.2. Isolate’s Identification and Antimicrobial Susceptibility Profiles

Collected isolates were identified using a phenotypic and genotypic method, VITEK2 automated system (bioMerieux, France) and polymerase chain reaction (PCR) amplification ofblaOXA-51 gene.Antimicrobial susceptibility testing was performed using the VITEK2 automated system (bioMerieux, France). Piperacillin + tazobactam (ptz), ceftazidime (caz), cefepime (fep), trimethoprim/sulfamethoxazole (sxt), gentamycin (cn10), ciprofloxacin (cip), cefotaxime (ctx), imipenem (imp), meropenem (mem), and tigecycline (tig) were tested.


## 2.3. Molecular Investigation of Resistance Mechanisms

### 2.3.1. Recovery of Isolates and Nucleic Acid Extraction

Recovery of a fresh pure colony on Muller–Hinton (MH) agar (Diagnostic Media Products, DMP, South Africa) was conducted from microbanks (Microbanks™, ProLab Diagnostics Inc., Canada) under aseptic environment. A single pure colony was used for DNA and RNA extraction. DNA was extracted using the boiling method [43]; briefly, a loopful of fresh pure colony was suspended in 1000 µL of saline (SABAX Pour Saline 0.9%, Adcock Ingram Critical Care (Pty) Ltd., South Africa) in an Eppendorf tube (Eppendorf AG, Hamburg, Germany) followed by incubation (Memmert Incubator IN30, Germany) for 20 min at 37°C ± 2. The suspension was then removed and well shaken using a vortex (Vortex, Heidolph, Reax top, Germany) at full speed for 20 s. Thereafter, the suspension was centrifuged (Mikro 20, Werk Nr. Bajahr E Kin, Hettich Zentrifugen, Germany) for 5 min at 130 × 100 rotation per minute (RPM). The supernatant was removed and resuspended in 200 µL of PCR grade water (BioConcept Ltd., Switzerland) in an Eppendorf tube (Eppendorf AG, Hamburg, Germany). The suspension was then put on a thermomixer (Eppendorf, Thermomixer Compact, MERCK, Merck Chemical (Pty) Ltd., South Africa) at 90°C for 10 mn at 700 RPM. Thereafter, it was put on a cell disruptor (Disruptor Genie®, Scientific Industries SI™, USA) for 10 min. Finally, the suspension was centrifuged (Mikro 20, Werk Nr. Bajahr E Kin, Hettich Zentrifugen, Germany) for 10 min at 130 × 100 RPM. The supernatant containing the DNA was transferred and preserved in the sterile Eppendorf tube (Eppendorf AG, Hamburg, Germany) and stored at −20°C.RNA extraction was conducted using a commercial RNA isolation kit (ISOLATE II RNA Mini Kit, MagMAX™ Viral/Pathogen, bioline, London, United Kingdom) following the manufacturer’s instructions [44]. Extracted RNA was quantified using a spectrophotometer (NanoDrop Lite Spectrophotometer, NanoDrop Products, Thermo Scientific, USA) and normalized to 1 μg/μL. The obtained RNA was stored at −80°C. A complementary DNA (cDNA) synthesis was performed using a cDNA synthesis kit (SensiFAST cDNA Synthesis Mix Reaction Guidelines, Bioline, a Meridian Life Science® Company, United Kingdom) following the manufacturer’s instructions. cDNA products were stored at −20°C, until further use.


### 2.3.2. Conventional PCR Amplification of AdeABC Efflux Pump (adeB, adeS, and adeR) and Quinolone (parC and gyrA) Resistance Genes

Gene amplification by conventional PCR was performed using 2X MyTaq HS Red Mix (MyTaq HS Red Mix, Meridian Bioscience, bioline, United Kingdom) to detect genes of interest inA. baumannii. The Bioline protocol [45] was followed to prepare multiplex PCR (M-PCR) assays using primers with similar melting temperatures and monoplex PCR (m-PCR) for primers of different annealing temperatures. To avoid mistakes during detection of amplicon by gel electrophoresis, the AdeABC efflux pump system associated genes adeB and adeS which differ by only 3 nucleotides in base pairs were ran separately. M-PCR was run for genes adeS and adeR and a m-PCR for gene adeB. PCR was performed in a reaction mixture of a total volume of 25 μL, composed as follows: 12.5 μL of 2X MyTaq HS Red Mix (MyTaq HS Red Mix, Meridian Bioscience, Bioline, United Kingdom), 0.5 μL of each primer, and 6.5 μL PCR grade water (BioConcept Ltd., Switzerland) were added to make up to 20 μL, and 5 μL of DNA template was added to constitute a 25 μL reaction mix. Negative and positive controls were run with every PCR on a thermocycler (GeneAmp® PCR System 2700, Applied Biosystems, Singapore). The thermocycling conditions for conventional PCR and primers used for detection of drug resistance-associated genes are detailed in Supplementary 2.


### 2.3.3. PCR Amplicon Detection

The detection of PCR amplicons was conducted in a 1% agarose gel stained with ethidium bromide.


### 2.3.4. Quantitative Real-Time PCR (qRT-PCR) Amplification of AdeABC Efflux Pump (adeB, adeS, and adeR)

The qRT-PCR was conducted on cDNA for the detection of the presence of mRNA, using the SYBR No-ROX kit (SensiFAST™ SYBR® NO-ROX Kit, Meridian Bioscience, Bioline, United Kingdom) on Sacace Real-Time PCR Instrument (SaCycler-96, Real-Time PCR System, Sacace Biotechnologies Srl, Scalabrini, Italy). The qRT-PCR reaction mixture was prepared in a volume of 20 μL comprising of 4 μL of cDNA, 10 μL SYBR, 0.8 μL of each primer, and 4.4 μL of PCR grade water (BioConcept Ltd., Switzerland). The thermocycling conditions and primers used are detailed in Supplementary 1 and 2, respectively.


## 2.3.1. Recovery of Isolates and Nucleic Acid Extraction

Recovery of a fresh pure colony on Muller–Hinton (MH) agar (Diagnostic Media Products, DMP, South Africa) was conducted from microbanks (Microbanks™, ProLab Diagnostics Inc., Canada) under aseptic environment. A single pure colony was used for DNA and RNA extraction. DNA was extracted using the boiling method [43]; briefly, a loopful of fresh pure colony was suspended in 1000 µL of saline (SABAX Pour Saline 0.9%, Adcock Ingram Critical Care (Pty) Ltd., South Africa) in an Eppendorf tube (Eppendorf AG, Hamburg, Germany) followed by incubation (Memmert Incubator IN30, Germany) for 20 min at 37°C ± 2. The suspension was then removed and well shaken using a vortex (Vortex, Heidolph, Reax top, Germany) at full speed for 20 s. Thereafter, the suspension was centrifuged (Mikro 20, Werk Nr. Bajahr E Kin, Hettich Zentrifugen, Germany) for 5 min at 130 × 100 rotation per minute (RPM). The supernatant was removed and resuspended in 200 µL of PCR grade water (BioConcept Ltd., Switzerland) in an Eppendorf tube (Eppendorf AG, Hamburg, Germany). The suspension was then put on a thermomixer (Eppendorf, Thermomixer Compact, MERCK, Merck Chemical (Pty) Ltd., South Africa) at 90°C for 10 mn at 700 RPM. Thereafter, it was put on a cell disruptor (Disruptor Genie®, Scientific Industries SI™, USA) for 10 min. Finally, the suspension was centrifuged (Mikro 20, Werk Nr. Bajahr E Kin, Hettich Zentrifugen, Germany) for 10 min at 130 × 100 RPM. The supernatant containing the DNA was transferred and preserved in the sterile Eppendorf tube (Eppendorf AG, Hamburg, Germany) and stored at −20°C.RNA extraction was conducted using a commercial RNA isolation kit (ISOLATE II RNA Mini Kit, MagMAX™ Viral/Pathogen, bioline, London, United Kingdom) following the manufacturer’s instructions [44]. Extracted RNA was quantified using a spectrophotometer (NanoDrop Lite Spectrophotometer, NanoDrop Products, Thermo Scientific, USA) and normalized to 1 μg/μL. The obtained RNA was stored at −80°C. A complementary DNA (cDNA) synthesis was performed using a cDNA synthesis kit (SensiFAST cDNA Synthesis Mix Reaction Guidelines, Bioline, a Meridian Life Science® Company, United Kingdom) following the manufacturer’s instructions. cDNA products were stored at −20°C, until further use.


## 2.3.2. Conventional PCR Amplification of AdeABC Efflux Pump (adeB, adeS, and adeR) and Quinolone (parC and gyrA) Resistance Genes

Gene amplification by conventional PCR was performed using 2X MyTaq HS Red Mix (MyTaq HS Red Mix, Meridian Bioscience, bioline, United Kingdom) to detect genes of interest inA. baumannii. The Bioline protocol [45] was followed to prepare multiplex PCR (M-PCR) assays using primers with similar melting temperatures and monoplex PCR (m-PCR) for primers of different annealing temperatures. To avoid mistakes during detection of amplicon by gel electrophoresis, the AdeABC efflux pump system associated genes adeB and adeS which differ by only 3 nucleotides in base pairs were ran separately. M-PCR was run for genes adeS and adeR and a m-PCR for gene adeB. PCR was performed in a reaction mixture of a total volume of 25 μL, composed as follows: 12.5 μL of 2X MyTaq HS Red Mix (MyTaq HS Red Mix, Meridian Bioscience, Bioline, United Kingdom), 0.5 μL of each primer, and 6.5 μL PCR grade water (BioConcept Ltd., Switzerland) were added to make up to 20 μL, and 5 μL of DNA template was added to constitute a 25 μL reaction mix. Negative and positive controls were run with every PCR on a thermocycler (GeneAmp® PCR System 2700, Applied Biosystems, Singapore). The thermocycling conditions for conventional PCR and primers used for detection of drug resistance-associated genes are detailed in Supplementary 2.


## 2.3.3. PCR Amplicon Detection

The detection of PCR amplicons was conducted in a 1% agarose gel stained with ethidium bromide.


## 2.3.4. Quantitative Real-Time PCR (qRT-PCR) Amplification of AdeABC Efflux Pump (adeB, adeS, and adeR)

The qRT-PCR was conducted on cDNA for the detection of the presence of mRNA, using the SYBR No-ROX kit (SensiFAST™ SYBR® NO-ROX Kit, Meridian Bioscience, Bioline, United Kingdom) on Sacace Real-Time PCR Instrument (SaCycler-96, Real-Time PCR System, Sacace Biotechnologies Srl, Scalabrini, Italy). The qRT-PCR reaction mixture was prepared in a volume of 20 μL comprising of 4 μL of cDNA, 10 μL SYBR, 0.8 μL of each primer, and 4.4 μL of PCR grade water (BioConcept Ltd., Switzerland). The thermocycling conditions and primers used are detailed in Supplementary 1 and 2, respectively.


## 2.4. Phenotypic Evaluation of AdeABC Efflux PumpadeB, adeS, and adeR Gene Expression

A functional AdeABC efflux system was assessed by evaluating the difference between the minimal inhibitory concentrations (MICs) for tigecycline (TGC) using the gradient diffusion method (tigecycline, MIC Test Strip, Liofilchem® Srl, Roseto d’Abruzzi, Italy) before and after exposure to an efflux pump inhibitor (EPI) carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Sigma-Aldrich, Dorset, United Kingdom) [46]. Briefly, CCCP was added to one of two MH agar plates at the final concentration of 100 µg/mL. From a fresh overnight culture of A. baumannii, a 0.5 Mac Farhland turbid sample was made (Densichek, BioMerieux DensiCHEK Plus, USA). A swab was then used to spread A. baumannii on each agar plate, followed by placement of a 0.016–256 mg/L TGC E-strip test in the middle of each agar plate followed by an overnight incubation at 37°C. E. coli strain ATCC 85218 was used as a control.


## 2.5. Sequencing of Quinolone Resistance-Associated Genes (parC and gyrA)

The Sanger sequencing method (ABI3500XL; Applied Biosymptoms, United States) at Inqaba Biotec (Pretoria) was used to sequence theparC and gyrA amplicons. Sequences were edited using ChromasPro software (version 2.0) and then aligned together with wild-type sequences from GenBank using BioEdit (http://www.mbio.ncsu.edu/BioEdit/BioEdit.html). Serine 83 of gyrA gene and serine 80 and serine 84 of parC gene were investigated for mutation. A phylogenetic tree was drawn using molecular evolutionary genetic analysis (MEGA) 4.0 [47] to evaluate evolutionary relatedness of the study strains.


## 2.6. Statistical Analysis

Data were captured on Microsoft Professional Excel 2016 and data analysis conducted on IBM SPSS Statistics 26 (IBM® SPSS® Statistics version 26.0, 2019). Ap value of ≤0.05 was considered statistically significant.


## 3. Results

### 3.1. Isolate Identification and Antimicrobial Susceptibility Profile

AllA. baumannii isolates identified by VITEK2 were PCR-positive for blaOXA-51. The antibiotic susceptibility of the collected isolates was tested against 10 commercially available antibiotics. The study isolates were 89% resistant to cefotaxime and 85% resistant to both ceftazidime and cefepime (Figure 1). The isolates were 76% resistant to piperacillin + tazobactam, a combination of penicillin and beta-lactamase inhibitor (Figure 1). Eighty-three percent (83%) of the isolates were resistant to imipenem and meropenem (Figure 1), while resistance to gentamycin, trimethoprim-sulfamethoxazole, and ciprofloxacin was 81%, 82%, and 83%, respectively (Figure 1). Antibiotic susceptibility testing for tigecycline showed that 87% of isolates were susceptible, while 3% demonstrated intermediate susceptibility (Figure 1).Figure 1
Antimicrobial susceptibility testing.


### 3.2. Molecular and Phenotypic Evaluation of the Active AdeABC Efflux Pump System as the Resistance Mechanism

The evaluation of an active AdeABC efflux pump system as a mechanism of resistance was conducted using a combination of genetic and phenotypic tests targetingadeB, adeR, and adeS genes. A positive PCR and qRT-PCR of targeted genes suggest that the genes of interest are present and that the related proteins are actively produced. The phenotypic evaluation confirms the actual use of the efflux pump system as the resistance mechanism at the phenotypic level.The positive PCR amplification of targeted genes was 100% foradeB and 99% for both adeR and adeS genes (Table 1; Supplementary 3). The qRT-PCR was 100% positive for adeB and 99% and 98.1% for adeR and adeS, respectively. A total of 100 isolates (97%) were positive for PCR and qRT-PCR targeting adeB, adeR, and adeS genes. Among the 100 isolates, 75% of these isolates phenotypically demonstrated the active use of efflux pumps as a drug resistance mechanism (Table 1; Supplementary 3) and 25% did not show any phenotypic level of efflux pump involvement in resistance to tigecycline. Of the 25% that did not demonstrate phenotypic expression of efflux pump use, 88% (22 isolates) had the complete required set of genes for an active efflux pump (Table 1; Supplementary 3). There was a statistically significant association between positive conventional PCR and qRT-PCR amplification of structural adeB and regulatory adeR genes and active phenotypic expression of the efflux pump (p value <0.05).Table 1
Combined analysis of molecular and phenotypic investigations of the active efflux pump.
Efflux pump patternNumber of samplesEfflux pump activeEfflux pump not activePCR B+R+S+1017526qPCR B+R+S+1007525PCR-qPCR B+R+S+1007525PCR, polymerase chain reaction; qPCR, quantitative polymerase chain reaction; +, positive PCR.


### 3.3. Investigation of Point Mutations inparC and gyrA

The point mutations inparC and gyrA genes were investigated following Sanger sequencing of PCR products. All study isolates (100%) were PCR-positive for gyrA and 99% positive for parC (Table 2; Figure 2; Supplementary 4), while 2 samples failed quality control for gyrA Sanger sequencing and could not be sequenced. The sequence analysis of gyrA revealed that 89% of the isolates showed a point mutation in serine 83 inducing a change in amino acid from serine to leucine (Table 2; Figure 3; Supplementary 4), while 11% did not have this mutation. All the isolates (100%) were negative for point mutation on serine 84 of parC gene; while on serine 80, 39% of isolates had a point mutation inducing change in amino acid from serine to leucine. This point mutation was not observed in 61% of the isolate sequences that were analysed (Table 2; Figure 3; Supplementary 4). The analysis of the two gene sequences revealed that 36% (37 isolates) had both point mutations. Serine 83 for gyrA and serine 80 for parC and 10 isolates did not have any of the mutations (Table 2; Supplementary 4).Table 2
Summary of mutation analysis inparC and gyrA genes.
Mutation in sequencegyrA +gyrA −TotalparC +37138parC −531063Total9011101There was no mutation in amplifiedparC sequence at serine 84; the data show only mutation at serine 80 for parC gene. +, presence of mutation inducing change in amino acid. −, absence of mutation inducing change in amino acid.Figure 2
Sequences analysis ofparC gene.
(a)(b)Figure 3
Sequences analysis ofgyrA gene.
(a)(b)The occurrence of mutations among isolates collected has increased over time. In 2017,parC mutation among collected isolates was 5% (1/22), 36% (14/32) in 2018, and 60% (25/42) in 2020 (Table 2; Figure 3; Supplementary 4). gyrA mutation in isolates showed a similar trend, 86% (19/22), 95% (37/39), and 85% (34/40) in 2017, 2018, and 2020, respectively (Table 2; Figure 3; Supplementary 4).


## 3.1. Isolate Identification and Antimicrobial Susceptibility Profile

AllA. baumannii isolates identified by VITEK2 were PCR-positive for blaOXA-51. The antibiotic susceptibility of the collected isolates was tested against 10 commercially available antibiotics. The study isolates were 89% resistant to cefotaxime and 85% resistant to both ceftazidime and cefepime (Figure 1). The isolates were 76% resistant to piperacillin + tazobactam, a combination of penicillin and beta-lactamase inhibitor (Figure 1). Eighty-three percent (83%) of the isolates were resistant to imipenem and meropenem (Figure 1), while resistance to gentamycin, trimethoprim-sulfamethoxazole, and ciprofloxacin was 81%, 82%, and 83%, respectively (Figure 1). Antibiotic susceptibility testing for tigecycline showed that 87% of isolates were susceptible, while 3% demonstrated intermediate susceptibility (Figure 1).Figure 1
Antimicrobial susceptibility testing.


## 3.2. Molecular and Phenotypic Evaluation of the Active AdeABC Efflux Pump System as the Resistance Mechanism

The evaluation of an active AdeABC efflux pump system as a mechanism of resistance was conducted using a combination of genetic and phenotypic tests targetingadeB, adeR, and adeS genes. A positive PCR and qRT-PCR of targeted genes suggest that the genes of interest are present and that the related proteins are actively produced. The phenotypic evaluation confirms the actual use of the efflux pump system as the resistance mechanism at the phenotypic level.The positive PCR amplification of targeted genes was 100% foradeB and 99% for both adeR and adeS genes (Table 1; Supplementary 3). The qRT-PCR was 100% positive for adeB and 99% and 98.1% for adeR and adeS, respectively. A total of 100 isolates (97%) were positive for PCR and qRT-PCR targeting adeB, adeR, and adeS genes. Among the 100 isolates, 75% of these isolates phenotypically demonstrated the active use of efflux pumps as a drug resistance mechanism (Table 1; Supplementary 3) and 25% did not show any phenotypic level of efflux pump involvement in resistance to tigecycline. Of the 25% that did not demonstrate phenotypic expression of efflux pump use, 88% (22 isolates) had the complete required set of genes for an active efflux pump (Table 1; Supplementary 3). There was a statistically significant association between positive conventional PCR and qRT-PCR amplification of structural adeB and regulatory adeR genes and active phenotypic expression of the efflux pump (p value <0.05).Table 1
Combined analysis of molecular and phenotypic investigations of the active efflux pump.
Efflux pump patternNumber of samplesEfflux pump activeEfflux pump not activePCR B+R+S+1017526qPCR B+R+S+1007525PCR-qPCR B+R+S+1007525PCR, polymerase chain reaction; qPCR, quantitative polymerase chain reaction; +, positive PCR.


## 3.3. Investigation of Point Mutations inparC and gyrA

The point mutations inparC and gyrA genes were investigated following Sanger sequencing of PCR products. All study isolates (100%) were PCR-positive for gyrA and 99% positive for parC (Table 2; Figure 2; Supplementary 4), while 2 samples failed quality control for gyrA Sanger sequencing and could not be sequenced. The sequence analysis of gyrA revealed that 89% of the isolates showed a point mutation in serine 83 inducing a change in amino acid from serine to leucine (Table 2; Figure 3; Supplementary 4), while 11% did not have this mutation. All the isolates (100%) were negative for point mutation on serine 84 of parC gene; while on serine 80, 39% of isolates had a point mutation inducing change in amino acid from serine to leucine. This point mutation was not observed in 61% of the isolate sequences that were analysed (Table 2; Figure 3; Supplementary 4). The analysis of the two gene sequences revealed that 36% (37 isolates) had both point mutations. Serine 83 for gyrA and serine 80 for parC and 10 isolates did not have any of the mutations (Table 2; Supplementary 4).Table 2
Summary of mutation analysis inparC and gyrA genes.
Mutation in sequencegyrA +gyrA −TotalparC +37138parC −531063Total9011101There was no mutation in amplifiedparC sequence at serine 84; the data show only mutation at serine 80 for parC gene. +, presence of mutation inducing change in amino acid. −, absence of mutation inducing change in amino acid.Figure 2
Sequences analysis ofparC gene.
(a)(b)Figure 3
Sequences analysis ofgyrA gene.
(a)(b)The occurrence of mutations among isolates collected has increased over time. In 2017,parC mutation among collected isolates was 5% (1/22), 36% (14/32) in 2018, and 60% (25/42) in 2020 (Table 2; Figure 3; Supplementary 4). gyrA mutation in isolates showed a similar trend, 86% (19/22), 95% (37/39), and 85% (34/40) in 2017, 2018, and 2020, respectively (Table 2; Figure 3; Supplementary 4).


## 4. Discussion

Acinetobacter baumannii increasing the spectrum of resistance to available antibiotics is of a public health concern [1]. Similar to previous studies, PCR amplification of blaOXA-51 gene was used as a genotypic identification and confirmatory method for A. baumannii strains previously identified by VITEK2 (bioMerieux, France) [49–51]. Even though it has been reported in literature that some of A. baumannii strains do not harbour blaOXA-51 [53], the gene has been reported intrinsic to the species by several authors [49–51, 53]. The use of blaOXA-51 has been recommended as a simple and reliable identification method for A. baumannii strains [49–51].This study results reveal a high resistance to most of available antibiotics used by clinicians in management ofA. baumannii infections (cefotaxime 89%, ceftazidime 85%, cefepime 85%, piperacillin + tazobactam 76%, imipenem 83%, meropenem 83%, ciprofloxacin 83%, gentamycin 81%, and trimethoprim-sulfamethoxazole 82%) (Figure 1). The findings are similar to several reports made by different researchers within the region [54–56] and globally [58]. In their published report on the district of Oliver Reginald Tambo in the Eastern Cape Province of South Africa, Anane et al. [55] highlighted that A. baumannii strains showed resistance rates above 80% against the same antibiotics tested in this current study. Similarly, Lowe et al. [57] in Tshwane district in Gauteng province of South Africa reported high prevalence of resistance (69–90%) of A. baumannii strains to the same antibiotics tested in the current study.An increasing number of isolates with an intermediate susceptibility to tigecycline (3%) is of concern (Figure1). A raise of resistance to last resort antibiotics against A. baumannii suggests that soon there will be no antibiotics with effective antibacterial action against locally circulating strains of A. baumannii. This report is another call to support cautious prescription and use of antibiotics in accordance with local and international guidelines [59] and stress the need to develop a new antimicrobial alternative against this bacterium.The evaluation of the AdeABC efflux pump system as a mechanism of resistance used byA. baumannii strains isolated at DGMTL revealed a high prevalence of adeB (100%), adeR (99%), and adeS (99%). This is in agreement with other studies that reported the AdeABC efflux pump system as the most prevalent and with the highest detection rate in clinical isolates compared to other efflux pump genes [26]. This may be explained by the physiologic role of the efflux pumps which are proteins on the bacterial cell membrane that regulate the movement of substances from the internal to the external cell environment [60]. However, a high prevalence of the AdeABC efflux pump in bacteria clinical isolates may be explained by the involvement of the pump in driving resistance against antibiotics as hypothesized by Ranjbar and his colleagues [61]. A study conducted in China demonstrated that the AdeABC efflux pump is responsible for an increase in multidrug-resistant A. baumannii strains in their paediatric intensive care unit [42]. Similar findings were reported in Iraq where the AdeABC efflux pump system was 96% prevalent in clinical isolates of A. baumannii [61]. Mahmoudi et al. [62] also mentioned that AdeABC efflux pump system genes were present in more than 90% of A. baumannii clinical isolates in their study. The positive statistical association of structural adeB and regulatory adeR genes may be explained by the molecular structural organisation of the AdeABC operon system regulated by adeRS. The adeR gene has been identified as a recognition response factor that acts as a transcriptional activator under the influence of adeS [26]. However, exploring the interaction between adeR and adeABC, Chang et al. [63] reported that adeR action can be independent of adeS influence. Their study revealed that adeR gene may be activated as a result of an amino acid substitutions in a repeat motif region between adeR and adeABC leading to adeABC overexpression and increase tolerance to antibiotic action. The complete understanding of the AdeABC efflux pump system working and regulation mechanism is still limited. Even though additional research is needed to explore the regulation of this mechanism of resistance, this report is inclined to support the finding by Chang et al. [63], whereby, despite the presence of adeS; some AdeABC efflux pumps system in the study isolates did not demonstrate phenotypic resistance to tigecycline (Table 1; Supplementary 3). This study data revealed that 97% (100/103) of the isolates had the required set of genes (adeB, adeR, and adeS) for expression of an active efflux pump (Table 1; Supplementary 3). Of these isolates, 75% (75/100) demonstrated an active use of the AdeABC efflux pump as a mechanism of resistance, while 25% (25/100) did not demonstrate at phenotypic level expression of the AdeABC efflux pump as a mechanism of resistance. Literature supports that it is not just the presence of the AdeABC efflux pump-associated gene but their overexpression triggered by a mutation that induce resistance to antibiotics [15, 63, 64]. Furthermore, it is documented that A. baumannii can use other efflux pump-associated genes [14, 65] and superfamily [22] to develop a multidrug-resistance profile. In addition, researchers reported that the AdeABC efflux pump need, in some cases, a synergistic action with other resistance mechanisms to express a level of resistance to particular antibiotic classes [67]. All these may explain the phenotypic result of 22/25 isolates of the study that did not demonstrate the use of the AdeABC efflux pump as an active mechanism of resistance, while the required set of genes was present, and their mRNA produced.Quinolone resistance-determining regions are specific regions on thegyrA and parC genes that code for the amino acid mutations that give A. baumannii its ability to resist quinolones [38, 39]. These quinolone resistance-determining regions are found on codon 80 (serine 80) and 84 (Glu-84) of the parC gene and codon 83 (serine 83) of the gyrA gene [40]. In this study, all the clinical isolates (100%) were PCR positive for gyrA and 99% positive for parC (Table 2; Figure 3; Supplementary 4). The sequencing analysis of gyrA revealed that 89% of isolates showed a point mutation in serine 83, 39.2% had mutation on serine 80, and none of isolates exhibits mutation on Glu-84 for parC-amplified gene. Several studies reported similar findings suggesting that amino acid substitutions on gyrA and parC genes are associated with quinolone resistance in A. baumannii [67, 68]. The phenotypic susceptibility testing of isolated strains of A. baumannii to ciprofloxacin (83% resistance) correlates with fluoroquinolone resistance-associated genes sequence analysis investigated in this study. However, it is known that other amino acid substitutions in gyrA induce A. baumannii resistance to quinolone. A study reported quinolone resistance mutations at serine 81 on the gyrA gene inducing change from serine to leucine [31]. Isolates showing mutations from glycine 81 to valine and alanine 84 to proline on the gyrA gene have also been reported to contribute to resistance to quinolone in A. baumannii [69, 70]. Of all the study isolates, 37 had mutations on both genes, while 10 did not have any mutations (Table 2; Figure 4; Supplementary 4). Presence of mutation in the both gyrA and parC gene contributes to a higher level of ciprofloxacin resistance rather than a single mutation in the gyrA or parC gene as reported by Lee and his colleagues [72]. The results of this current study support the report by Lee et al. [72]. The 37 isolates with mutation on both genes revealed higher level resistance to tigecycline than the 10 isolates that did not have any (Table 2; Supplementary 4). These results suggest that single or double mutation on parC and/or gyrA and their concomitant presence or absence on both genes affect the level of resistance to antibiotics.Figure 4
(a) Phylogenetic analysis of the gyrA gene sequences showing highly related strains. The evolutionary history was inferred using the neighbour-joining method [48]. (b) Phylogenetic analysis of the parC gene sequences showing highly related strains. The evolutionary history was inferred using the neighbour-joining method [48].
(a)(b)The phylogenetic relatedness of the isolates was investigated using the neighbour-joining method [48] on MEGA 4.0 (Figures 4(a) and 4(b)). The analysis showed very minimal evolutionary distance between the isolates from DGMAH. This observation is most likely because of the nature of the genes; parC and gyrA are housekeeping genes very essential for replication and thus highly conserved.This study has also observed that mutations within theA. baumannii isolates have gradually increased over time from 2017 to 2020, implying that isolates at DGMAH are increasingly becoming resistant to drugs used to treat them. Other studies have shown a gradual increase in resistance in healthcare institutions [57, 73, 74].


## 5. Conclusion

Isolates ofA. baumannii at DGMAH demonstrated a high resistance prevalence to available antibiotics. The use of the AdeABC efflux pump system as an active mechanism of resistance combined with point mutation mainly in gyrA contributes to broaden the resistance spectrum of A. baumannii isolates at DGMAH. This situation is particularly alarming as the locally isolated strains demonstrated an increase in resistance to tigecycline. Judicial use of antimicrobials supported by antibiotic susceptibility results should be instituted to control the rise of resistance in A. baumannii strains.



---

*Source: 9923816-2021-10-07.xml*