Isolating and Diagnosing Burkholderia Sppfrom Soil and Soil Surrounding Onion Roots in Najaf Governorate

Abstract

The present study included the isolation and identification of Burkholderiaspp. samples were collected from places near soil and associated with plant onion roots ;rice farms;palm trees; pinus;Frangula alnus and Medic ago sativa)in soil AL-Najaf province. All the samples were collected  During a four- month  from October 2020 to March 2021.  Burkholderia  isolates were isolated from(90) environmental sample from (soil ).The isolates were identify according to morphology  properties , cultural, conventional biochemical test then the diagnosis was confirmed by using Vitek 2 GN-card .Burkholderia spp isolates The  isolates were obtained from soil  and soil surrounding the onion root (Rhizospheres) for different  plants group were S₄1(2.2)℅and S⁵1(2.2)℅ .The researcher  reached to that DNA sequencing is the best method for identification of bacteria and used of the best medium to growth bacteria it'sOxidation Fermentation Polymyxin Bacitracin Lactose Agar Basecompared with   used medium other.         

         PCRtechnique was used for the detection and amplification of (recA) gene in twoBurkholderia spp.isolates and the results showed that all tested isolates harbor this gene, and analysis was investigated for confirming the identification of  two selective Burkholderia spp.isolates understudy. PCR product from the amplification of 16S rRNA gene were send for DNA Sanger Sequencing to Macrogen Company / Korea. The results of gene amplification and sequencing successfully confirm the identification and showed that two isolates belonged to Burkholderia spp.bacteria in similarity ratios (95)% with the Burkholderia spp.isolates in Gen Bank. After sequencing analysis, two isolates were deposited inGenBank was the two types species Mohammad(1)and Mohammad(2)were first recorded in Iraq, in Najaf province .

Burkholderia spp. isolate(S₄,S₅)was resistant towards Azithromycin , Augmentinand Amikacin (100%), while (S₅) sensitivity of isolates towards clavulanic acid, Meropenem and Cefazolin .While(S₄)sensitivityof isolates towards Tobramycin and Ciprofloxacin, Cefepime ; Ceftazidime.

Finally, the virulence factor of  Burkholderia sp. all of the isolates were able to produce Gelatinase enzyme and biofilm isolates were tested. Positive results are demonstrated by partial liquefaction of the inoculated tube andunability to produce urease enzyme.The aim of study  isolation and identification of BurkholderiaSp.from different environmental sources ,determination the bio patterns of isolates using Vitek 2 system and PCR.,Molecular identification using diagnostic primers and using specialized primers to identify virulence factors for environmental and studyof ten antibiotics and their effect on bacterial isolates and Determine the tolerance capacity  of bacteria  with study  internal growth factors  such as Temperature and PH.

In the molecular study, five genes related to virulence factors and resistant antibiotic were detected, namely PRND, OPCI,  CciI,  Bla-tem  and PltC. Environmental samples possessed genes for the production of antifungal and antibacterial and the formation of purine proteins,and This explains the adaption Bacteria by habitat type in which they reside