The Expression of pmr A gene in Acinetobacter baumannii Bacteria that Responsible for Colistin Resistance

A total of Forty isolates of Acinetobacter baumannii was Collected from many Hospitals (Teaching Baghdad Hospital, Teaching Pediatric Hospital, Al-Shahid Kazy Al-Harery and teaching clinical laboratories, Al-Yarmok hospital and Al-Kadhimia hospital) in Baghdad at the period 15/11/2018 to 19/2/2019, including sputum 15 isolates (37.5%), Blood 14 isolates (53%), wound swab 4 isolates (15%), Urine 4 isolates (10%), Burns one isolate (2.5%), pleural effusion fluids one isolate (2.5%) and throat swab one isolate (2.5%) . The current study showed that six isolates of A. baumannii that were resistant to colistin (polymyxin) carry the ( pmrA ) gene at product size was 175bp. The gene expression of the pmr A gene for bacteria was measured once it was treated with a Colistin (polymyxin) antibiotic at a concentration (<=0.5µl) of 0.48 ml and without treated by using MIC (minimum inhibitory concentration) method as it was measured. It was observed that there was no increase in the gene expression of pmrA gene after the bacterial isolate was treated with Colistin (polymyxin

Colistin (polymyxin) is an important antibiotic in the treatment of infection caused by the highly multidrug resistant MDR-mediated bacterium including A. baumannii, which produces the carbapenimase enzyme [4].
With the frequent use of Colistin in the treatment of infection, A. baumannii bacteria were resistant to Colistin [5]. In Gram-negative bacteria, resistance against Colistin (polymyxin) is acquired through changes in target sites for Colistin that targets (lipopolysaccharide LPS), as there are two types of resistance mechanisms against colistin [6]. First, inhibition of lipid A (which is the main component of lipopolysaccharide) in Gram-negative bacteria, which leads to loss of LPS, which is the target of the antibiotic (Colistin) mutations in the (pmrA) and PM gene stimulate. The pmrC gene that adds the (Phospho-ethanolamine) group (PEtn) to (Heptaacylated) for lipid A as mutations in these two genes (pmrAB) activate the second mechanism of resistance [7].
The aims of study is to isolate of Acinetobacter baumannii from different pathologies sources and to the express the gene of pmrA which that resistance genes.   [9]. Steps of PCR reaction in Table (1). After that 5μL of the PCR product was transferred to the agarose gel electrophoresis at a concentration 2% with Ethidium bromide (0.5) µg/ ml, as well as loaded 5μl of DNA Ladder (100) bp. at (100) volts for (80) minutes. The gel was examined using a UV-Transilluminator (300 nm) [10].

Gene expression by using Real Time PCR
The kit was used (One Step RT-qPCR) manufactured by (Promega, USA). The mix reaction of Real time PCR in Table (2)  The lowest percentage of isolates of bacteria from burns samples came from only one isolate at a rate of 2.5% of the total number of isolates and an average of one isolate from chest fluids and one isolate from throat swabs at a rate of (2.5%) for each of them. These results are consistent with [17] as the lowest percentage of isolates was from body fluids, which amounted to (10) isolates from a total of (111) isolates belonging to A. baumannii at a rate of (9%). This result is in agreement with the [12] as the lowest percentage of bacteria isolation from throat swabs samples was (8%). The current study showed that only six isolates of A.
baumannii that were resistant to colistin (polymyxin). The results of our study showed that only six isolates that are resistant to colistin carry the (pmrA) gene at percentage (15%) the bands are equal in product size 175bp as shown in figure (1). The researchers [8] in their study they measured the gene expression of the pmrA gene in A.

Conclusions
The current study showed that all isolates of A. baumannii that were resistant to colistin (polymyxin) carry the (pmrA) gene the bands are equal in product size 175bp. While there was no increase in pmrA gene expression after treatment bacteria with Colistin antibiotic.