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Molecular detection of genomic islands responsible for antimicrobial resistance in Acinetobacter baumannii strain A92
Other Title(s)
التحري الجزيئي للجزر الوراثية المسؤولة عن المقاومة للمضادات الحياتية في العزلة Acinetobacter baumannii A92
Dissertant
Thesis advisor
Adhiah, Ali Husayn
Melconian, Alice Krikor
University
University of Baghdad
Faculty
College of Science
Department
Biotechnology Department
University Country
Iraq
Degree
Ph.D.
Degree Date
2014
English Abstract
The present study aimed to detect and map a novel comM gene-associated genomic island (GEI) in strain A92 of Acinetobacter baumannii, which is called A.
baumannii resistance island (AbaR).
In addition, tRIP (tRNA site Interrogation for Pathogenicity)-PCR analysis was employed to examine eight A.
baumannii strains (A92, A473, A424, A457, A14, AYE, A25 and ACICU) for the presence or absence of GEIs at two integration hotspot loci (#7 and #24).
Furthermore, biofilm and pellicle formation was in vitro phenotypically screened in nine clinical strains of A.
baumannii (A473, A92, AYE, A424, A457, ATCC, A14, A25 and ACICU).
Finally, copA (copper-resistance associated) gene present in the GEI of A.
baumannii A92 strain was cloned and characterized.
Before carrying out the forthcoming assessments, antimicrobial susceptibility was tested and revealed that the A.
baumannii A92 strain was sensitive to imipenem and pipracillin-tazobactam; intermediate to tobramycin; and resistance to amikacin, ceftazidime, ciprofloxacin, gentamycin and minocycline.
After that, A novel AbaR1-like genomic island in strain A92 of A.
baumannii was detected by PCR-mapping, which revealed a putative region of about 37254 bp by designing sets of primers for each 10 Kb segment of the region 56540-93793 bp by using the NCBI (National Center for Biotechnology Information) sequence data (GenBank accession number AMGF01000002.1) of A.
baumannii IS-116 strain.
The results of PCR-mapping for the first 10 Kb (56540-66285 bp) on gel electrophoresis gave bands of different molecular size (4037, 1023, 613, 4503 and 8136 bp).
Mapping of the second 10 kb sequence segment (region 66285-73261) revealed that the amplified region was presented with two bands (384 and 7319 bp).
In the third amplified 10 Kb segment (region 72181-83041 bp), two bands (242 and 7005 bp) were detected.
During mapping of the latter region, some primers pairs did not amplify this region; therefore, a two-step chromosomal Summary ===================================================================== ii walking method was applied by using two primers (sequencing primer and chromosomal walking primer) to determine what type of sequence that was present in this region.
Agarose gel electrophoresis showed a band with a molecular size of 150 bp for the walking primer and R-IS-1164/5 pair, and sequence analysis confirmed the presence of copA gene when it was screened in BLAST (Basic Local Alignment Search Tool).
During the detection of AbaR1-like island in A.
baumannii A92 strain, the location of some transposition genes (tniE and tniA) were determined by PCRmapping, which revealed a band for tniE gene (734 bp) and a band for tniA gene (714 bp).
After that and to determine the 3' end of the GEI under study, the two step chromosomal walking method was employed further to determine the 3'-comM end of the putative AbaR1-like island by using a known region of tinA gene.
The results of PCR product sequencing confirmed the presence of 3'-end of comM gene when screened in BLAST, and there was 94% sequence-homology to the competence protein of comM gene in more than one type of five A.
baumannii strains (BJAB0715, A424, D1279779, AB307-0294 and AB0057).
With respect to tRIP-PCR analysis, the results revealed that most of the eight investigated A.
baumannii strains had an amplicon (GEI is not disrupted ) in locus #7, with the exception of A457 strain that showed a negative result (no amplicon), which means that the #7 locus of A457 strain was disrupted by a GEI.
Much more negative results were observed in locus #24, in which six strains (A92, A473, A424, A457, A14 and AYE) showed no amplicons, which mean a presence of foreign GEIs.
Strains A25 and ACICU were an exception, in which positive results were obtained.
Biofilm formation was performed after 24 hour incubation time period at two temperatures (30°C and 37°C) for the nine clinical strains of A.
baumannii, and after reading the absorbance at 600 nm, a variation in biofilm formation among the Summary ===================================================================== iii investigated strains was observed, while the results of pellicle formation for these strains revealed that only A92 strain had the ability to form pellicle at 25°C, which appeared as a white layer on the surface of Muller-Hinton broth.
Finally, cloning of copA that was present in the novel GEI of A.
baumannii A92 was performed after designing two primers that contained two restriction sites for XhoI (CTCGAG) and KpnI (GGTACC) by using PWSK29 as a cloning vector, which was isolated from E.
coli DH5α.
Agarose gel electrophoresis of the PCR product indicated the presence of copA gene in the AbaR genomic island, which appeared as a band with a molecular size of 2424 bp.
Then, after extraction of copA gene and vector from the gel, they were double digested with KpnI and XohI enzymes, and after their purification, the digested vector and insert (copA) were religated, and after that, the ligation mixture was transformed into E.
coli DH5α competence cells.
The transformed E.
coli DH5α were cultured overnight on Luria agar plates that contain X-gal and ampicillin, which aid in detecting bacterial cells that contain recombinant vector that appeared as white coloured colonies instead of a blue colour.
To ensure that the PWSK29 vector has the desired gene, colony PCR for white colonies was performed by using two primers (copA-R primer and M13-F as universal primer).
The results confirmed the presence of the insert gene, which appeared as band with a molecular size of 2484 bp after agarose gel electrophoresis.
Main Subjects
Topics
No. of Pages
119
Table of Contents
Table of contents.
Abstract.
Abstract in Arabic.
Introduction.
Chapter One : Literature review.
Chapter Two : Materials and methods.
Chapter Three : Results and discussions.
Conclusions and recommendations.
References.
American Psychological Association (APA)
al-Ujayli, Suhad Sad Mahmud. (2014). Molecular detection of genomic islands responsible for antimicrobial resistance in Acinetobacter baumannii strain A92. (Doctoral dissertations Theses and Dissertations Master). University of Baghdad, Iraq
https://search.emarefa.net/detail/BIM-601468
Modern Language Association (MLA)
al-Ujayli, Suhad Sad Mahmud. Molecular detection of genomic islands responsible for antimicrobial resistance in Acinetobacter baumannii strain A92. (Doctoral dissertations Theses and Dissertations Master). University of Baghdad. (2014).
https://search.emarefa.net/detail/BIM-601468
American Medical Association (AMA)
al-Ujayli, Suhad Sad Mahmud. (2014). Molecular detection of genomic islands responsible for antimicrobial resistance in Acinetobacter baumannii strain A92. (Doctoral dissertations Theses and Dissertations Master). University of Baghdad, Iraq
https://search.emarefa.net/detail/BIM-601468
Language
English
Data Type
Arab Theses
Record ID
BIM-601468