الكشف الجزيئي عن جيني LecA و LasA واختبار فعالية بعض المستخلصات النباتية ضد بكتريا الزوائف الزنجارية المعزولة محلياً

Molecular detection of LecA and LasA genes and antibacterial activity of some plant extracts against locally isolated Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa found in most natural environments (soil, water, plants and on the skin). It is distinguished by its color between yellow, green and blue and its smell that resembles the smell of grapes or fruits. It is a gram-negative, aerobic, opportunistic, pathogenic Bacillus that causes diseases in humans and animals. When these bacteria enter wounds, or people who are immunocompromised (burn patients “the most prominent example”), they generally cause severe infections, sepsis and tissue destruction. And if it hits vital body systems such as the lungs, urinary tract or kidneys, it may lead to death.

Ninety samples were collected from clinical samples (61 from burn patients) and 29 environmental samples including soil and water. Identification of Pseudomonas aeruginosa isolates were carried out by using biochemical tests. Further these isolates identities were confirmed by PCR technique to amplify LecA and LasA genes.
An antibiotic sensitivity test was performed using a number of antibiotics using the VITEK device for 24 isolates, 8 of which are environmental and 16 clinical, these isolates were selected according to the diagnostic ratio of the VITEK device.
The isolates for which the sensitivity test was performed showed very high resistance to the antibiotics Ampicillin, Amoxicillin, Cefuroxime, Meropenem it was 100% . The resistance rate for Amikacin and Gentamicin was (72.7%) and (88.8%) respectively, while all isolates were sensitive for Colistin (100%), and then suggested that colistin was the most effective antibiotic.
The results of the electrophoresis of the polymerase chain reaction of LecA gene for 24 isolates (5 soil, 3 water, 16 clinical) selected because of their high antibiotic resistance showed that 19 isolate( environmental isolates and 11 clinical isolates) possess the gene and showed bands in agarose gel electrophoresis with a size of 369 base pairs.
As for the LasA gene, the results of the electrophoresis of the polymerase chain reaction of for 24 isolates (5 soil, 3 water, 16 clinical) selected because of their high antibiotic resistance showed that 18 isolate (environmental isolates and 10 clinical isolates) possess the gene and showed bands in agarose gel electrophoresis with a size of 226 base pairs.
Then, ten sample of PCR products were sent to Macrogen company(Korea) for the sequencing, programs such as Mega and Blast were used to compare the obtained isolates with global isolates(NCBI), the results of comparing the obtained isolates with global isolates of the LecA gene showed that two isolates were close to previous studies, while the remaining three were new strains. As for the results of the LasA gene, it was shown that one isolate is close to previous studies, and four isolates were new strains.
Six plant extracts were extracted by methanol, ethanol and hot water were tested, namely (Cinnamon, Pimpinella anisum, Nigella sativa, Artemisia, Myrtus communis and Quercus cortex.), and tested against three isolates from different sources (clinical, soil and water), where the first five of these plant extracts showed no antibacterial activity against P. aeruginosa, while Quercus cortex was effective, as the inhibition zone was clear when tested with four concentrations (25, 50, 75 , 100)mg\ml.