Role of opr D gene in biofilm formation and imipenem resistance in pseudomoanas aeruginosa

العناوين الأخرى

دور جين oprDمقاومة الاميبينيم و تكوين الغشاء الحياتي في بكتريا Pseudomonas aeruginosa

مقدم أطروحة جامعية

Musafir, Hadil Karim

مشرف أطروحة جامعية

al-Mathkhuri, Harith Jabbar Fahd

أعضاء اللجنة

al-Hassani, Hayfa Hadi
Muruj, Abd al-Sattar
al-Qazzaz, Abd al-Karim A.
Yusuf, Hana Salim
Isa, Rajwa Hasan

الجامعة

جامعة بغداد

الكلية

كلية العلوم

القسم الأكاديمي

قسم علوم الحياة

دولة الجامعة

العراق

الدرجة العلمية

دكتوراه

تاريخ الدرجة العلمية

2013

الملخص العربي

Fifty eight Pseudomonas aeruginosa strains evaluated with E test; forty seven (81.03%), two (3.4٥%), and nine (15.5۲%) strains were susceptible, intermediate, and resistant to imipenem, respectively.

All the resistant and intermediate susceptible strains, previously isolated from patients with cystic fibrosis, showed no carbapenemase activity as confirmed by the Hodge test.

The relationship between biofilm formation, imipenem resistance, and oprD expression were assessed in some imipenem resistant clinical P.

aeruginosa strains (SMC631C, SMC631F, SMC631J, SMC631H, SMC631K and SMC 4974).

All resistant strains showed low oprD expression and low biofilm values in comparison to the sensitive ones.

The results in this study was revealed that oprD::isphoA/hah resistant strain developed significantly lower biofilm formation capacity than PAO1 wild type.

Moreover, this mutation triggered Imipenem resistance (P < 0.001 by comparison to PAO1 strain).

During this work, an strain with point mutation (SMC631F-ImR) in oprD gene was obtained.

This mutant was resistant to imipenem (MIC> 32 mg/l) and weak biofilm former (OD550= 0.06) compared with the parent SMC631 biofilm (OD550=0.19).

Expression of poprD::isphoA/hah plasmid in the oprD mutant fully restores the biofilm defect observed for the oprD mutant.

There were no significant differences (P>0.05) between PAO1 and complemented strain ; nevertheless, significant differences (P<0.05) were noticed between the complemented strain and the mutant oprD::isphoA/hah with vector.

urthermore, poprD::isphoA/hah plasmid expression restored imipenem sensitivity (4.3) to the level of wild type and there were no significant difference (P>0.05) between them.

However, significant differences were found between the complemented strain and the mutant oprD::isphoA/hah with empty vector.

Similar results were obtained with the clinical strain SMC631.

An SMC631F-ImR/poprD plasmid introduced into the oprD mutant.

Again, expression of SMC631F-ImR / poprD+ in the oprD mutant fully restores the biofilm defect observed for the oprD mutant relative to the empty vector as control.

Taking together, these results confirm that the inactivation of oprD is responsible for the observed defect of biofilm phenotype.

mariner transposon mutagenesis was performed in two clinical strains comprised the highest biofilm former.

Additionally, biofilm deficient strains were screened by microtiter plate assay, in the mutant derivatives (originated from SMC576 and SMC214).

We mapped four different genes, pilY1, pilW, pslI and algC.

The results revealed that pilY::Mar mutants and pilW::Mar19 mutant showed significantly deficient biofilm formation in comparison to the parent SMC576, pilY1::Mar represented five independent mutant strains (five insertion sites in pilY1 gene) and one strain with insertion in pilW gene.

Moreover, mutation of pilY1 and pilW in the SMC576 background lead to loss of twitching motility.

The swarm phenotypes of SMC576 mutants were shown.

The pilY1::Mar mutants and pilW::Mar19 mutant exhibit a pattern of swarming motility in comparison with SMC576 (weak swarm, shorter and fewer tendrils).

The second highest biofilm is formed by SMC214; were mapped two different genes pilW, pilX I that responsible for biofilm deficient.

Those biofilms formed by the pilX::Mar and pilW::Mar mutants were significantly lower (P < 0.001) than the biofilm of the SMC214 parent strain.

The pilW::Mar and pilX::Mar mutants exhibit swarming phenotypes that closely resembled the SMC214 parent strain; while the pilW::Mar and pilX::Mar showed strong suppressor of twitching motility.

We introduced constructed plasmid, +ppilY1 plasmid into the pilY1::Mir8 mutant, pilY1:Mir8 expression restored the higher biofilm formation to the levels comparable to the SMC576.

Furthermore, the expression of +ppilY1 in the pilY1::Mir8 mutant fully restored the swarming defect observed for the pilY1::Mir8 mutant relative to the vector control.

Regarding twitching motility, the expression of PilY1 was observed to complement the twitching defect of the pilY1::Mir8 mutant.

Collectively, these results confirmed the inactivation of pilY1 alone which responsible for the observed suppression of SMC576 mutant phenotypes.

The map of mutants pslI::Mar and algC::Mar showed the differences in Psl that produced by pslI::Mar and algC::Mar mutants which were highly deficient biofilm.

التخصصات الرئيسية

الأحياء

الموضوعات

• علم البيئة
• الكائنات الدقيقة
• الأغشية الحيوية

قائمة المحتويات

• APA
• MLA
• AMA

لغة النص

الإنجليزية

نوع البيانات

رسائل جامعية

رقم السجل

BIM-598730