Studying the Gene Expressions of Bla OXA-51Like and Bla OXA-23Like in Acinetobacter baumannii

Genotypic detection of some Antibiotics Resistant genes by polymerase chain reaction (PCR) (20): isolates of Acinetobacter baumannii that showed resistance to Ceftaxim, Cefotaxim, Cefepim, and Imipenim were selected. The results showed that 20 isolates of A. baumannii possess the bla-OXA23-like gene and that all isolates possess this gene with a percentage of 100% and a molecular weight of 605 bp. The current study showed that A. baumannii isolates carry 100% of the bla-OXA51like gene when studied with 20 isolates that are resistant to antibiotics (Imipenim, Ceftazidime, Cifepime, and Cifexime) that belong to this group of β-lactame with a molecular weight of 382 bp. Gene expression was done for the bla-OXA23 like and bla-OXA51 like. The results of gene expression for the bla-OXA23 like gene show a decrease in gene expression when treated with Ceftazidime.

The bla-OXA23like gene encodes for OXAs enzymes (a gene prevalent among A. baumannii) [4], and in another study conducted in Lebanon, it was shown that 76.5% of this gene was isolated among isolates of A. baumannii resistant to anti-carbapenim [5].In another study in India, it was shown that the OXA-51 enzyme, which is encoded by the bla-OXA51-like gene, was isolated from 103 carbapenem-resistant bacterial isolates.This type of enzyme is resistant to penicillins and cephalosporins, as well as cephalosporinase inhibitors, including clavulani acid [6].

Collection of sample:-
A total of 40 isolates of A. baumannii were obtained from many hospitals in Baghdad, including the Teaching Baghdad Hospital, Pediatric Hospital, AL Shahid Kazy, AL Harery, and teaching clinical laboratories, AL-Yarmok Hospital, and AL Kadhimia Hospital, from the period of November 15, 2018 to February 19, 2019, including sputum 15 (38%), blood 14 (35%), Urine sample (4%), wound swab 4 (10%), pleural effusion fluid, Throat swab and Burn one isolates (2.5%) each of them, Clinical samples were cultured in different media (blood agar, MacConkey agar, and CHROM agar) and incubated at 37°C for 24 h.For diagnosis, we used biochemical tests, including oxidase and catalase, and microscopic examination; for the final detection of isolates, we used the Vitik 2 compact system (France) and the API 20E test (France) [7].

Genetic detection of the bla OXA-23 like and bla OXA-51-like gene
The sequence (Primers) that was used to detect bla OXA-23 like (F: TCTGGTTGTACGGTTCAGC) (R: AGTCTTTCCAAAAATTTTG) 650 bp and bla OXA-51-like (F:ACAGAARTATTTAAGTGGG) (R:GGTCTACAKCCMWTCC CCA) 500 bp antibiotic resistance genes in a total volume of (25) µl including the forward (1)µl and reverse (1)µl, (2)µl of DNA template, (8.5)µl of deionized-free water (Go Taq master mix) with 12.5 µl.After the PCR products were prepared, they were mixed with a Vortex and placed in the PCR.A thermalcycling system depending on the conditions and temperatures of the bla OXA-23 like and bla OXA- 51-like genes optimum [8].
The steps of the PCR reaction were: Step 1 (only one cycle for 5 minutes at a temperature of 95°C for the primary DNA denaturation); Step 2 (30 cycles included: A: 30 sec at 95°C for DNA template denaturation; B: 30 sec at 53°C for the primers to bind to DNA template annealing; C: 40 sec at 72°C for the associated primers to be elongated.Step3 (only one cycle for 7 minutes at 72 °C is needed for the final elongation of the double DNA strip

Agarose Gel Electrophoresis
After PCR amplification, 5μL of the product of the PCR was transferred to electrophoresis on an agarose gel at a concentration of 2% with Ethidium bromide (0.5) µg/ ml.(5) µl of the PCR product was transferred to the pits designated for it, as well as loaded 5μl of DNA ladder (100 bp) at a voltage of 100 volts for 80 minutes.The gel was examined by using a UV-Transilluminator (300 nm).[9]

Real Time PCR and gene expression
As a result of real-time PCR) experiments, the kit was used (one-step RT-qPCR) manufactured by (Promega, USA, as shown in Table 1.The steps of real-time PCR were: Step 1 (only one cycle for 15 minutes) at a temperature of 37°C for cDNA and for the initial denaturation of DNA (5 minutes) at a temperature of 95°C, Step 2 (40 cycles included): A: 20 sec at 95°C for DNA template denaturation; B: 20 sec at 53 °C for the primers to bind to DNA template annealing; C: 30 sec. at 72°C for the associated primers to be elongated.Step 3 (three cycles for one sec.at 72°C to 95°C for the melted green Calculate the amount of change in the level of gene expression as shown by the following equations: Folding =2 -ΔΔCT ΔΔCT =ΔCT Treated -ΔCT Control ΔCT =CT gene -CT House Keeping gene (16SrRNA)

Detection of bla-OXA23 like gene
Twenty isolates of Acinetobacter baumannii that showed resistance to (Ceftaxim, Cefotaxim, Cefepim, and Imipenim) belonging to the β-lactam group were selected.The results showed that 20 isolates of A. baumannii possess the bla-OXA23-like gene, which encodes for the enzyme (Carbapenimase that is responsible for resistance to β-lactam antibiotics, and that all isolates of A. baumannii possess this gene at a percentage (100%).with a molecular weight of 605 bp, as shown in Figure 1 and Table 2. Several studies indicated that many genes that encode carbapenemase enzymes that are related to resistance to anti-carbapenems (Imipenim, Meropenim).The resistance of bacteria to these antibiotics occurs due to their production of βlactamase enzymes that degrade beta-lactams, and this leads to the resistance of A. baumannii to antibiotics.OXA23-like with a percentage (95.4%)[11] Whereas [12] indicated that A. baumannii isolated from different clinical cases carries the bla-OXA23like gene at a rate of 97.7%.The reason is that this gene has a high prevalence among clinical bacterial isolates.

Detection of bla-OXA51 like gene:-
The current study showed that A. baumannii isolates carry 100% of the bla-OXA51like gene when studied with 20 isolates that are resistant to antibiotics (Imipenem, Ceftazidime, Cifepime, Cifexime) that belong to this group of β-lactam.bla-OXA51 like gene encoding enzymes Carbapenemase.When comparing the doubled bundles with the ladder, it had a molecular weight of 382 bp, as shown in Figure 2 and Table 3.These results were in agreement with the study conducted by [11], where the percentage of the presence of the bla-OXA51like gene was 91%, and this was also indicated by researchers [13] when they found that the percentage of the presence of the gene bla -OXA 51 like) in isolates of A. baumannii was 91.3%).In another study conducted by [14], they showed that the presence of the gene bla-OXA51ike), which encodes for Carbapenimase enzymes, amounted to 100%, which is a result consistent with what we have found in the current study.No. of isolate Antibiotics that are bacteria resistant to beta-lactam group bla-OXA51like 1

Gene expression measurement of the bla-OXA23 LIKE gene
The gene expression of the anti-β-lactam-resistant bla-OXA23like gene was also measured by quantitative polymerase chain reaction (real-time PCR), as the reaction curve was running well with no contaminants, as shown in Figure 3. Primers for the gene (bla-OXA23 and 16S rRNA) were used using the SYBR green method.
Gene expression measurement of the bla-OXA23like gene of A. baumannii resistant to betalactam antibiotics after it was treated once with the anti-Ceftazedim (0.19 ml/L depending on the MIC concentration).It was noted that there was no increase in gene expression when the bacterial isolate was treated with the antibiotic (Ceftazedim), folding was measured depending on (2 -ΔΔCT ) and again in the absence of the antibiotic (Ceftazedim) as it was (folding based on 2 - ΔΔCT ) (1.00), which was considered (control positive) as shown in Table 4.

Gene expression measurement of the bla-51OXA like gene
The gene expression of the β-lactam-resistant bla-OXA51like gene was measured by quantitative polymerase chain reaction (Real time PCR) using primers for the gene (bla-OXA51 and 16S rRNA on the SYBR green method).The reaction curve was running well with no pollutants, as shown in Figure 4.
Measuring the gene expression (bla-OXA51 like) of a bacterial isolate of A. baumannii resistant to beta-lactam after treatment with ceftazedim at a concentration of 0.19 ml/L depending on the (MIC) concentration, as (folding) was measured based on 2 -ΔΔCT and again in the absence of the antibiotic (Ceftazedim), where (folding) was based on 2 -ΔΔCT (1.00) counted as (Control positive) as shown in Table 5.An increase in gene expression was observed when the bacterial isolate was treated with the antibiotic Ceftazedim.

Conclusion
The gene expression for the bla-OXA23like gene showed a decrease when treated with Ceftazidim, whereas the bla-OXA51 like gene, showed an increase in gene expression when treated with Ceftazidime.

Figure 1 .
Figure 1.Electrophoresis on agarose gel (2%) of bla oxa 23 like gene amplifiers of A. baumannii at a voltage of 100 V/cm for 80 minutes

Figure 2 . 3 .
Figure 2. Electrophoresis on agarose gel (2%) of bla oxa 51 like gene amplifiers of A. baumannii at a voltage of 100 V/cm for 80 minutes Table 3. Resistance of bacteria to beta-lactams because they possess the bla-OXA-51 like gene.

Figure 3 .
Figure 3. Quantitative polymerase chain reaction curve for bla-OXA23 gene, the violet color represents the sample treated with the antibiotic Ceftazedim, the green color represents the sample without treatment.

Figure 4 .
Figure 4. Quantitative polymerase chain reaction curve for bla-OXA51 gene, blue color represents the sample treated with the antibiotic Ceftazedim, the red color represents the sample without treatment.

Table 1 .
A result of gene expression (bla OXA-23 like and bla OXA-51-like) by using RT-PCR

Table 4 .
Gene expression values of 16SrRNA and bla-OXA23 like

Table 5 .
Gene expression values for 16srRNA and bla-OXA51 like gene