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Table of Contents
ORIGINAL ARTICLE
Year : 2022  |  Volume : 21  |  Issue : 1  |  Page : 29-36

Identification and sequencing of ISAba2 of Acinetobacter baumannii isolated from baghdad hospitals


1 College of Science, 2012, Baghdad University, Baghdad, Iraq
2 Department of Microbiology, College of Medicine, Mustansiriyah University, Baghdad, Iraq
3 Department of Gene Bank, Forensic DNA for Research and Training Centre, Al-Nahrain University, Baghdad, Iraq

Date of Submission21-Aug-2021
Date of Decision17-Oct-2021
Date of Acceptance25-Oct-2021
Date of Web Publication30-Jun-2022

Correspondence Address:
Mr. Salah Sabah Muhsin
B.Sc. of Biology, College of Science, Baghdad University 2012, Baghdad
Iraq
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mj.mj_17_21

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  Abstract 

Background: The ability of multidrug-resistant Acinetobacter baumannii to survive in any situation including the acquisition of many different kinds of virulence factors and antibiotic resistance genes is the primary source of worry in hospital settings. Despite their low hydrolysis capability, oxacillinase (OXA) types are often associated with genetic factors such as insertion sequences (ISs) in order to enhance carbapenemase production and mobilization. As a result, assessing the frequency of IS genes in A. baumannii is extremely essential in many hospitals and medical institutions. Aims: The goal of this work is to find the IS ISAba2, which may have a role in antibiotic resistance in extensively drug-resistant (XDR) A. baumannii. Methods: The polymerase chain reaction (PCR) was used to confirm the presence of A. baumannii by identifying the blaOXA-51 gene. According to the current Clinical and Laboratory Standards Institute recommendations (2020), antimicrobials are determined using the Kirby–Bauer disc diffusion technique on Mueller-Hinton agar. The molecular research of ISAba2 includes PCR and Sanger sequencing of the PCR results. Results: Among 38 A. baumannii isolates, 23 (61%) and 25 (66%) were resistant to meropenem and imipenem, respectively. The blaOXA-51 gene was detected in all 21 XDR strains tested; furthermore, ISAba2 was found in all 21 XDR-analyzed A. baumannii isolates. Conclusions: ISAba2 has a high predominance between extreme drug-resistant A. baumannii. The identification of these parameters can assist in the control of infection and decrease of the microorganism's prevalence rate.

Keywords: Insertion sequences, mobile genetic elements, multidrug resistant (extensively drug resistant)


How to cite this article:
Muhsin SS, Bakir WA, Sabbah MA. Identification and sequencing of ISAba2 of Acinetobacter baumannii isolated from baghdad hospitals. Mustansiriya Med J 2022;21:29-36

How to cite this URL:
Muhsin SS, Bakir WA, Sabbah MA. Identification and sequencing of ISAba2 of Acinetobacter baumannii isolated from baghdad hospitals. Mustansiriya Med J [serial online] 2022 [cited 2022 Dec 2];21:29-36. Available from: https://www.mmjonweb.org/text.asp?2022/21/1/29/349305


  Introduction Top


Acinetobacter baumannii is a common opportunistic pathogen among immunocompromised persons, especially those who have remained more than 90 days in the hospital.[1] Commonly associated with aquatic environments.[2] It has been recognized as a “red alert” human pathogen, causing concern among medical professionals due to its wide range of antibiotic resistance.[3]

The development of multidrug-resistant (MDR) microorganisms has become a major concern in the context of both nosocomial and community-acquired illnesses.[4] According to the World Health Organization, antibiotic resistance is one of the three most serious challenges confronting human health today.[5] The therapeutic options are restricted, which frequently leads to ineffective treatment and unfavorable effects for patients.[6]

Enzymatic (production of β-lactamases and enzymatic modification of aminoglycosides) and nonenzymatic processes (changes in membrane permeability, efflux pump activation, and target site modification) are the major mechanisms of antibiotic resistance.[7]

In all species, insertion sequences (ISs) constitute basic mobile genetic elements (MGEs). They may travel horizontally across genomes as part of other MGE vectors such as phages and plasmids, or vertically inside a genome as part of other MGE vectors such as phages and plasmids (between different DNA molecules or within a specific DNA molecule).[8],[9] So far, approximately 4500 ISs from 29 families have been found.[9] Resistance is aided by the insertional inactivation of proteins such as transcriptional regulators and outer membrane proteins. In Acinetobacter spp., more than 30 IS types have been discovered, confirming that ISs are significant in the development of this species and have contributed to MDR phenotypes.[10]

ISs are short DNA sequences that act as simple transposable elements. ISs have two different features, and they are short (up to 2500 bp). Insertion Sequences have two different features, are short (up to 2500 bp) and only code for proteins that are involved in the transposition process. The transposase, which catalyzes the enzymatic process that allows the IS to transfer, and one regulatory protein, which either enhances or suppresses the transposition activity, are generally the proteins involved.[11]

IS ISAba2 is associated with resistance genes blaOXA-58 and blaAmpC.[12] Therefore, determining the prevalence of ISAba2 in A. baumannii in different hospitals and medical centers is very important.


  Methods Top


Specimen collection

A total of 540 different clinical specimens were collected during the period between October 2020 and February 2021 including sputum, blood, fluids (cerebrospinal, pleural, and peritoneal), urine, and swabs from five hospitals in Baghdad including the Burn Teaching Hospital, the Martyr Ghazi Al-Hariri Hospital, Baghdad Teaching Hospital, Welfare Teaching Hospital in the Medical City, and Al-Yarmouk Teaching Hospital.

Isolation of bacteria

In the laboratory (Teaching Laboratories/Medical City, Baghdad), the obtained specimens were cultured directly on blood agar and MacConkey agar under aseptic circumstances and incubated for 24 h at 37°C. The nonhemolytic opaque creamy colonies on blood agar and nonlactose-fermenting colonies on MacConkey agar were subcultured on another MacConkey agar plate and incubated for another 24 h at 37°C to obtain pure well-isolated colonies.[13]

Bacterial identification

Microscopical examination

One isolated colony was transferred to a microscopic slide, fixed, and then stained with Gram stain. Gram reaction, cell shape, and arrangement were recorded. The results were compared with Brooks et al.[14]

Biochemical tests

Biochemical assays such as growth at 42°C, culture on selective media, negative oxidase test, absence of lactose fermentation, and others were used to phenotypically identify the isolates as A. baumannii.

Identification of Acinetobacter baumannii using VITEK® 2 system

Bacterial isolates were identified at species level by using VITEK® 2 system with Identification-Gram Negative Bacteria (ID-GN) cards according to the manufacturer's instructions. A single colony of the bacterial isolates (previously incubated at 37°C for overnight on MacConky agar plates) were taken then suspended in 0.45% sodium chloride. The turbidity of bacterial suspension was measured to match the McFarland (0.5) standard, then the (Gram Negative VITEK® 2 ID card) with the bacterial suspension tubes were loaded manually into the VITEK® 2 system, the software also prepared according to (BioMerieux, France) the manufacturer's instructions.[15]

Identification of Acinetobacter baumannii by polymerase chain reaction

Polymerase chain reaction (PCR) was done to amplify blaOXA-51-like genes, which was used for the identification of A. baumannii isolates by using a primer specific for blaOXA-51-like genes.

Antimicrobial susceptibility test

A panel of antimicrobial susceptibility testing of the isolates to 18 agents was determined by Kirby–Bauer disc diffusion method on Mueller-Hinton agar (Oxoid/England) according to the current Clinical and Laboratory Standards Institute guidelines (2020).[16] The antibiotic discs (Mast Group/UK) used throughout the study for A. baumannii isolates are listed in [Table 1].
Table 1: Sequences of primers used throughout the study

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Extraction of genomic DNA from bacterial isolates

DNA was extracted from all bacterial isolates under study using a commercial purification system ZR Fungal/Bacterial DNA MiniPrep Kit designed for the isolation of DNA from Gram-negative bacteria according to the manufacturer's instructions. A total of 4 μL of DNA extract was used for each reaction.

Molecular recognition of blaOXA-51-like gene and insertion sequence elements

For the detection of extensively drug-resistant (XDR) A. baumannii isolates, PCR was used for identification of the blaOXA-51-like gene and ISAba2 elements. Primer sequences for each gene reported above are listed in [Table 1]. These primers (Macrogen, South Korea) were received in lyophilized state, dissolved in sterile deionized distilled water to a final concentration of 100 picomole/μl, and kept in a deep freezing until use, as advised by the vendor.

The PCR amplification procedure for the genetic level to detect genes under study included the following steps: final volume for PCR mixture was 25 μl (12.5 μl of Green Master Mix 2x, 4 μl extracted template DNA, 1.5 μl from each forward and reverse primer, 5.5 μl nuclease-free water) were added in 0.2 ml PCR Eppendorf tubes, mixed for a short time via vortex then have been loaded to Veriti™ 96-Well (Applied Biosystems) thermal cycler. The program used for each monoplex PCR reaction was set according to each primer. The best annealing temperature was chosen after the gradient runs through the optimization process of each oligonucleotide primer.

For amplification of blaOXA-51-like gene and ISAba2, the DNA thermal cycler device Veriti™ 96-Well (Applied Biosystems) was programmed in the following amplification conditions: following a 5-min activation at 94°C, 40 cycles of 45 s at 94°C (denaturation), 56°C (annealing), and 45 s at 72°C (extension) were conducted. The last cycle was followed by a 7-min period at 72°C [Table 2]. Amplified PCR products were examined by 1.5% agarose gel at an electrical current of 7 volt\cm2 in 1X TBE buffer with added RedSafe dye (iNtRON Biotechnology/Korea) has been exposed till the tincture reached the other side of the gel. The SiZer™-1000 Plus DNA Marker and SiZer™-100 DNA Marker (iNtRON Biotechnology/Korea) were used as a marker during PCR products electrophoresis. Thereafter, the agarose gel was removed from the tank and visualized by ultraviolet transilluminator documentation system (Cleaver Scientific/UK) with 336 nm and then photographed using a digital camera.
Table 2: Programs were used in the polymerase chain reaction for oxacillinase-51 and ISaba2 genes

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Sequencing was carried out by Macrogen DNA Sequencing (Seoul, Korea) using 3730xl DNA Analyzer (Applied Biosystems™, Foster City, CA). One specimen of ISAba2 PCR products with forward primer (17 pmol/μl) for the gene was selected and sent to sequencing. Raw reads generated in this study were trimmed or filtered to remove low-quality sequences using (SnapGene software). Once sequencing reads have been obtained, the data analysis process was started. The Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI) was used to analyze DNA sequences and do similarity searches.

Statistical analysis

The IBM SPSS Statistics 24 (IBM Corp., Armonk, NY) and Microsoft® Excel® 2019 MSO software were used to analyze the data. The Chi-square test was used to establish a significant comparison between percentages (0.05 and 0.01 probability in this research).


  Results Top


A total of 540 different clinical specimens were collected between October 2020 and November 2021 from some hospitals in Baghdad city including Baghdad Medical City and Al-Yarmouk Hospital. Preliminary identification of isolates is done through cultural identification, microscopic examination, and biochemical tests. All nonlactose fermenter isolates were also identified at species level with VITEK® 2 system by using ID-GNB cards, and the results showed that isolates under study belong to A. baumannii. Thirty-eight bacterial isolates were collected, all of which were identified as A. baumannii, as revealed by [Figure 1].
Figure 1: Frequency of clinical specimens that give positive result of Acinetobacter baumannii from all isolated specimens

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Finally, all the 21 XDR A. baumannii isolates were also identified depending on a blaOXA-51-like genes (carbapenem resistance genes), and all isolates gave a positive result.

Thirty-eight bacterial isolates were obtained from various clinical specimens including 12 isolates (32%) from sputum, 11 (29%) swabs, 8 (21%) urine, 5 (13%) blood, and 2 (5%) fluid, as shown in [Figure 2].
Figure 2: Distribution of Acinetobacter baumannii isolates according to type of positive specimen from total 38 positive isolates of Acinetobacter baumannii

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The gender distribution of the infected patients with A. baumannii is summarized in [Figure 3]. The incidence was higher among males 58% than that of females 42%. [Table 3] shows the distribution of A. baumannii Isolates according to the source and gender.
Figure 3: Distribution of Acinetobacter baumannii isolates according to gender

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Table 3: Distribution of Acinetobacter baumannii isolates according to the source and gender

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An assessment was then made of the relative numbers of A. baumannii isolates according to the type of specimen with respect to gender. Males differ significantly in terms of the number of A. baumannii in either specimen (P = 0.0001). However, in the female group, the differences were also highly significant (P = 0.0001), as illustrated in [Table 3].

By observing the results in [Table 4], A. baumannii show a high level of resistance of clinical isolates to most of the antibiotics under test. In this study, antibiotic resistance profile of A. baumannii for 18 antibiotics as follows: trimethoprim/sulfamethoxazole 32 (84%), ciprofloxacin 27 (71%), piperacillin/tazobactam 26 (68%), ticarcillin/clavulanate 26 (68%), ceftazidime 26 (68%), ceftriaxone 26 (68%), cefotaxime 25 (66%), imipenem 25 (66%), doxycycline 26 (68%), levofloxacin 25 (66%), cefepime 24 (63%), tetracycline 24 (63%), meropenem 23 (61%), gentamicin 22 (58%), amikacin 17 (45%), tobramycin 15 (39%), ampicillin/sulbactam 12 (32%), and colistin 0 (0%).
Table 4: Frequency and percentage of resistance Acinetobacter baumannii against antimicrobial agents

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Resistance of A. baumannii clinical isolates to the tested antibiotics showed that from 38 isolates, 32 (84%) were resistant to more than at least one antibiotic in three or more than three antibiotic groups. Consequently, it was crucial to classify all A. baumannii isolates into two major categories including non-MDR (N-MDR) and MDR, MDR classified into three categories including MDR, XDR, and pandrug resistant (PDR). Classification was done according to the criteria mentioned by Magiorakos et al.[17] Thus, all 38 A. baumannii isolates were classified as MDR in the first place; however, the majority of them were converted to be XDR or even PDR depending on their resistance profile and as follows: there were only six isolates (16%) of A. baumannii isolates that were considered as N-MDR since they were susceptible to all antimicrobial categories but resistant to some, not all agents listed in the extended-spectrum cephalosporin category. There were 32 isolates (84%) of A. baumannii isolates that were classified as MDR since they were resistant to ≥3 out of all antimicrobial categories that match the criteria used. It should be noted that even the isolate could resist to at least one agent in three or more antimicrobial categories; it would be considered resistant isolate to that category. There were 21 isolates (66%) of A. baumannii isolates that were considered as XDR since they were susceptible to only 2 out of all antimicrobial categories that match the criteria used. Thus, XDR was defined as nonsusceptibility to at least one agent in all but remained susceptible to only one or two antimicrobial categories. There were no isolates that show PDR pattern.

Genomic DNA was successfully extracted from all bacterial isolates by using genomic DNA purification kit, and the DNA bands were confirmed and analyzed by gel electrophoresis. Then, the purity of extracted DNA was determined between 1.7 and 2.

The 21 XDR A. baumannii isolates were identified to species level depending on carbapenem-hydrolyzing genes (blaOXA-51-like) (oxacillinases [OXAs]) belonging to class D β-lactamase. Each DNA extracted sample was subjected to a monoplex PCR reaction with a primer of this gene. All isolates gave a positive result, and the PCR products have been confirmed by comparing its base-pair size (434 bp) with SiZer-100 DNA Marker and SiZer™-1,000 plus DNA Marker, as shown in [Figure 4]. One of the present study objectives is to detect the existence and prevalence of ISAba2 genes. Each DNA extracted sample was subjected to monoplex PCR reaction with specific primer sets of ISAba2 genes. The PCR products have been confirmed by comparing its base-pair size (372 bp) with SiZer-100 DNA ladder, the results presented in [Table 5], showed that the presence of ISAba2 gene in all (100%) XDR A. baumannii isolates, as shown in [Figure 5].
Figure 4: Detection of blaOXA-51-like gene by monoplex polymerase chain reaction for Acinetobacter baumannii isolates. Lanes 1-21, XDR Acinetobacter baumannii; Lane C, Negative control. Lane M, 100 bp DNA marker. Lane N, 1000 bp plus DNA marker. Detection was done on agarose gel (1.5%) at 5 V/cm for 1.5 h, stained with RedSafe dye, and visualized on a ultraviolet transilluminator documentation system

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Figure 5: Detection of ISAba2 gene by monoplex polymerase chain reaction of isolates. Lanes 1-21, XDR Acinetobacter baumannii isolates. Lane C, Negative control. Lane M, 100 bp DNA ladder. Detection was done on agarose gel (1.5%) at 5 V/cm for 1.5 h, stained with RedSafe dye, and visualized on a ultraviolet transilluminator documentation system

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Table 5: Number and percentage of ISAba2 genes for all extensively drug-resistant Acinetobacter baumannii clinical isolates

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Monoplex PCR products for genes under study were detected by agarose gel electrophoresis and then the sequencing was carried out by (Macrogen Research, Seoul, Korea). Aligning of the obtained sequences with those of reference strains in GenBank confirmed the correct identification of these genes by PCR at (99%) for sequenced strand. The sequence was analyzed by BLAST software in NCBI. [Figure 6] shows the alignment result with Indian isolate (Accession number (CP050415.1). The sequence was found to share 99% nucleotide homology with the reference isolates. [Table 6] and [Table 7] show the nucleotide changes and features of ISAba2 (Forward) from A. baumannii (X7 isolate) with A. baumannii strain PM193665 chromosome from India.
Figure 6: Sequence alignment of ISAba2 (forward) of Acinetobacter baumannii (X7 isolate) with Acinetobacter baumannii strain PM193665 chromosome from India. Sequence ID: CP050415.1, length: 3948262. Range 1: 3611018 to 3611308

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Table 6: Nucleotide changes of ISAba2 (forward) from Acinetobacter baumannii (X7 isolate) with Acinetobacter baumannii strain PM193665 chromosome from India

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Table 7: Features of ISAba2 (forward) from Acinetobacter baumannii (X7 isolate) with Acinetobacter baumannii strain PM193665 chromosome, from India

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  Discussion Top


A. baumannii has developed as a well-established nosocomial pathogen with a high degree of antibiotic resistance. Due to rising resistance rates, carbapenems, the medication of choice for treating A. baumannii infections, are becoming more ineffective.[18] Carbapenem resistance is often linked to the development of OXA enzymes. Metallo-β-lactamases, on the other hand, may make A. baumannii resistant to carbapenems.[19]

This study out of 540 different clinical specimens shows that the highest isolation was from sputum and swab specimens more than other sources, followed by urine specimens, and the low percentage was in the blood and fluid specimens. In a local study, 170 bacterial isolates were obtained from various clinical specimens including 53 isolates (31.18%) from sputum, 45 (26.47%) wound, 43 (25.29%) blood, 12 (7.06%) burn, 10 (5.88%) urine, 5 (2.94%) fluid, and 2 (1.18%) ear swab.[20]

Another study in Iraq revealed that A. baumannii was isolated in high percentage 44 (38.26%) from sputum specimens; while, blood specimens constituted 30 (26.09%), wound specimens achieved 27 (23.48%), urine specimens formed 8 (6.95%), and the low percentage was in burn specimens which accomplished 6 (5.22%).[21] The variations in isolation percentage of pathogenic bacteria in studies may be attributed to differences in many factors such as sanitary practices for hospital staff, environmental conditions, isolation and identification techniques, social and cultural level of patients, differences in the prevalence of infection from one country to another, and difference in patients, which may inhibit or stimulate the growth and distribution of bacteria in hospitals.[22]

The incidence of infection was higher among males than that of females. These results agree with another study.[23]

According to this study, significantly high resistance rates to Trimethoprim/sulphamethoxazole and Ciprofloxacin, and moderate resistance to Piperacillin/tazobactam, Ticarcillin/clavulanate, Ceftazidime, Ceftriaxone, Doxycycline, Cefotaxime, Levofloxacin, Cefepime, Tetracycline, and Gentamicin. Low resistance rates were recorded to Amikacin, Tobramycin, and Ampicillin/sulbactam and no resistance to Colistin (0%). The important findings of the current study indicated that the 38 A. baumannii isolates were moderately resistant to meropenem and imipenem. On the other hand, the significantly high degree of sensitivity rates to tobramycin 50% and colistin 100% w detected.

The findings of this study corroborated the findings of previous studies on A. baumannii resistance to the majority of antibiotics, indicating that these bacteria have a wide range of resistance mechanisms. This would imply that there are significant risks among hospitalized patients, particularly where this antibiotic class was previously thought to be the usual therapy for A. baumannii infections.[24]

Results of resistance of A. baumannii clinical isolates to the tested antibiotics showed a high percentage of MDR isolates, while few isolates were N-MDR. Furthermore, a high number of isolates were XDR and no isolate was PDR. These results agreed with another study found that from 102 isolates, 95 (93.14%) isolates were resistant to more than two classes of antibiotics (MDR).[25] Thus, bacterial isolates that are characterized as XDR will also be characterized as MDR. Similarly, bacterial isolates would have to be XDR for them to be further defined as PDR. The recognition of the blaOXA-51-like gene can be utilized to identify A. baumannii simply and reliably.[26],[27]

Furthermore, because this gene was controlled by ISs such as ISAba1, the presence of intrinsic chromosomally placed genes of the blaOXA-51-like gene did not correlate with the amount of carbapenem resistance of A. baumannii isolates.[28] The results of the current study revealed that blaOXA-51-like genes are found in all 21 studied XDR A. baumannii isolates. The findings of this study corroborated those of previous local investigations and revealed that all 127 A. baumannii isolates were positive for blaOXA-51-like gene.[20] Another local study discovered that the blaOXA-51-like gene is present in all 96 isolates diagnosed as A. baumannii based on PCR data.[29] Another study in Egypt found that genes encoding for blaOXA-51 belonging to class D carbapenemases were found in 100% of the isolates.[30]

The results of this study revealed that all 21 (100%) XDR isolates carry the ISAba2 gene. A study in Iran found that the prevalence rate of ISAba2 in five hospitals was 92.38%, and in three of them (Taleghani, Milad, and Mofid), the prevalence rate was 100%,[23] but in Egypt, found that the prevalence of ISAba2 was 2.7%.[30]


  Conclusions Top


A. baumannii is a significant pathogen in several nations. According to the findings of this study, it has a high resistance rate against most antibiotics threatening inpatients as a red alarm bacterium in hospitals, producing a high rate of death and morbidity due to its numerous mechanisms of resistance and the fact that it is not or only rarely treated with conventional antibiotics.

This bacterium can cause dangerous and long-term infections, especially in youngsters and people with immunological deficiencies. Our research focused on certain mobile components that can be transported between species to change the antimicrobial pattern and enhance antimicrobial resistance. ISAba2 has a high prevalence among extreme drug-resistant A. baumannii isolated from several Baghdad hospitals. The identification of these parameters can aid in the control of infection and the reduction of the microorganism's prevalence rate.

Recommendations

Whole genome sequencing of Acinetobacter baumannii to determine the number and distribution of ISAba2 in the whole genome. Study the effects of ISAba2 insertion on the antibiotic resistance in A. baumannii. Determine the relationship between ISAba2 with β-lactamase encoding genes. Study the amino acid variation of ISAba2 in local A. baumannii isolates in comparison to reference isolates, as well as the effect on the resultant protein.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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Abouelfetouh A, Torky AS, Aboulmagd E. Phenotypic and genotypic characterization of carbapenem-resistant Acinetobacter baumannii isolates from Egypt. Antimicrob Resist Infect Control 2019;8:185.  Back to cited text no. 30
    


    Figures

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