Molecular detection of genomic islands responsible for antimicrobial resistance in Acinetobacter baumannii strain A92

العناوين الأخرى

التحري الجزيئي للجزر الوراثية المسؤولة عن المقاومة للمضادات الحياتية في العزلة Acinetobacter baumannii A92

مقدم أطروحة جامعية

al-Ujayli, Suhad Sad Mahmud

مشرف أطروحة جامعية

Adhiah, Ali Husayn
Melconian, Alice Krikor

الجامعة

جامعة بغداد

الكلية

كلية العلوم

القسم الأكاديمي

قسم التقانة الإحيائية

دولة الجامعة

العراق

الدرجة العلمية

دكتوراه

تاريخ الدرجة العلمية

2014

الملخص الإنجليزي

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.

التخصصات الرئيسية

الأحياء

الموضوعات

• الأدوية
• البكتيريا

عدد الصفحات

119

قائمة المحتويات

• APA
• MLA
• AMA

لغة النص

الإنجليزية

نوع البيانات

رسائل جامعية

رقم السجل

BIM-601468