Detection of the effect of synthetic siRNA on efflux pump MexA gene expression and antibiotic resistance in clinical isolates of Pseudomonas aeruginosa

Introduction and Aim: Pseudomonas aeruginosa is a nosocomial infection with an ability to develop high levels of antibiotic resistance. The efflux pump system is one of the mechanisms that is linked to multidrug resistance in P. aeruginosa . In this study, we employed siRNA loaded on gold nanoparticles against the MexA efflux pump gene to decrease the MexA gene expression in P. aeruginosa and estimated antibiotic resistance after gene silencing. Materials and Methods : This study examined four strains of P. aeruginosa isolated from patients in various hospitals in Baghdad. Bacteria isolated were identified by biochemical tests and Vitek compact 2 system. Single-stranded siRNA (33bp) designed in this study was loaded onto gold nanoparticles (AuNPls). Detection of the MexA gene was carried out by conventional PCR technique. The expression of MexA gene was examined by qRT-PCR in order to determine if the siRNA have impacted on MexA gene expression and on the antibiotic resistance in P. aeruginosa Results: This study showed that the mRNA expression level of the MexA gene exhibited a decrease in fold change ΔΔCT -2 in P. aeruginosa (isolates numbers 67, 66,49, and PDR(5p)) when examined in vitro . The specific fold change values observed were (0.202, 0.040, 0.063, and 0.163) respectively. The resistance percentages of antibiotics tested was observed to increase after MexA gene silencing. Conclusion: Targeting the MexA gene with synthetic siRNA may be a unique approach to diminish P. aeruginosa resistance to antibiotics. However, many unexpected consequences may occur when utilizing any genetic manipulation in bacteria


INTRODUCTION
seudomonas aeruginosa is a Gram-negative opportunistic pathogen associated with severe infections in community and healthcare settings (1).P. aeruginosa is considered a public health threat, as the pathogen displays high drug resistance to commonly used antimicrobials, making it difficult to treat infections caused by this bacterium (2,3) due to harboring many virulence factors (4).
P. aeruginosa drug resistance mechanism is considered multifactorial, one of them being efflux pumps that are responsible for transport of substrates across bacterial membranes (5).
The efflux pumps in P. aeruginosa are divided into five families: Resistance nodulation cell division (RND) families, major facilitator subfamily (MFS), small multidrug regulator (SMR), energy-dependent ATPdriven pumps are the ATP-binding cassette (ABC) family, and multidrug and toxic compound extrusion (MATE) (6).Among the efflux pumps, the RND efflux system MexAB-OprM is found to be strongly associated with its intrinsic resistance to most antimicrobial agents (7,8).The MexAB-OprM system includes the membrane proteins MexA, MexB, OprM, and MexR.MexA serves as a link between OprM and MexB.MexB mediates the expulsion of particular antibiotics based on their selective recognition while, OprM a lipoprotein is responsible for mediating the antibiotic's ultimate expulsion across the outer membrane.MexR auto regulates MexR and functions as a repressor of the MexAB-OprM system (9).Recent studies have demonstrated the RNA interference (RNAi) strategy to effectively silence gene expression in several bacterial species (10,11).The Small interfering RNA (siRNA) which are double stranded RNA of length ~18-25 bp, mediate post-transcriptional gene silencing of target genes and knocking down gene expression in various target cells (12).Studies with pathogens have shown siRNAs to efficiently alter virulence and drug resistance in these bacteria (13,14) and suggested as a probable alternative treatment option in pathogens that are multidrug resistant.The purpose of this study was to design and synthesize a siRNA targeting MexA, a component of the MexAB-OprM efflux pump in P. aeruginosa, and investigate into its potential to silence the expression of MexA gene and its influence on antibiotic susceptibility of P. aeruginosa.

MATERIALS AND METHODS
The study protocol and the subject information and consent form were reviewed and approved by a committee on publication ethics at the Institute of Genetic Engineering and Biotechnology, Baghdad, Iraq (No.1314 B dated 15 th May 2022).

Bacterial isolates and identification
Pseudomonas aeruginosa was isolated from clinical samples procured from hospitals in Baghdad.Identification of P. aeruginosa was based on morphology and biochemical parameters including oxidase, catalase, Triple Sugar Iron, and Indole tests (15).Confirmation of P. aeruginosa identification was done using the Vitek-2 compact system (Biomerieux, France).

DNA extraction and PCR detection of MexA gene
DNA was extracted from P. aeruginosa isolates using a PrestoTM Mini gDNA Bacteria Kit (Geneaid, Taiwan) according to manufacturer's instructions.Amplification of the efflux pump MexA gene was done using the PCR assay performed using the EasyTaq® PCR SuperMix kit (TransGen, China).Specific primers were designed in this study for the amplification of a 583bp of the MexA gene, the sequences of which are Forward primer: 5' GCAGACGGTGACCCTGAATA-3' and Reverse primer: 5'-GT ATTGGCTA CCGTCCTCCA-3'.The PCR mixture (25 μl) consisted of 2×EasyTaq® PCR SuperMix (12.5 μl); Template DNA (3 μl); Forward and reverse Primers (1 μl each of 10 pmol/μl primer) and nuclease free water (7.5 μl).The PCR was carried out in a thermal cycler with cycling conditions for 32 cycles being: Initial denaturation at 95 o C for 5mins; denaturation at 95 o C for 45 secs; annealing at 58 o C for 45 secs; extension at 72 o C for 45 secs followed by a final extension at 72ºC for 5 mins.The PCR products were examined by agarose gel electrophoresis on 1% agarose stained with ethidium bromide (Himedia, India), and visualized using UV transilluminator.A DNA ladder of 100bp (TransGen, China) was used as a reference to compare the size of DNA fragments.

SiRNA design and synthesis
In this study, using bioinformatics tools a 33bp single strand siRNA sequence 5'-AACGCCAGCCATGCGT GTACTGGTTCCGGCCCT-3' against MexA gene of P. aeruginosa was designed and outsourced for synthesizing by Macrogen, Korea.

Antibacterial effect of gold nanoparticles against P. aeruginosa
The minimum inhibitory concentration (MIC) of gold nanoparticles for the survivability of bacterial isolates was determined based on prior research (17) with slight modifications.The detection of the antibacterial effect of gold nanoparticles was carried out by microtiter plate method using Resazurin dye as follows: overnight cultures of P. aeruginosa were diluted approximately to 10 4 CFU/ml.Different concentrations (100, 50, 25, 12.5, 6.5, 3, 1.5, 0.75, 0.38, and 0.18 ppm) of gold nanoparticle (5-20nm, Vira carbon nanomaterials, Iran) solution were prepared by mixing with nutrient broth in the ratio of 1:2.To each well of the microtitre plate, 100 μl of gold nanoparticle suspension, 100μl of diluted P. aeruginosa culture and 20 μl of Resazurin dye (Sigma, USA) was added, mixed thoroughly and incubated overnight at 37°C.The antibacterial effect of gold nanoparticles on the bacteria was assessed visually by noting the color change from pink (viable) to blue (nonviable).

Conjugating siRNA onto gold nanoparticles (AuNPs)
Initially, a mixture of AuNP (100 ppm) was prepared by mixing 3ml of AuNP with 1ml of 3M NaCl.Serial dilutions of this mixture were prepared using different concentrations of NaCl.In order to facilitate the binding and loading of the siRNA onto the surface AuNPs, 20 µl of (100 pmol/l) siRNA was added to 4 ml of AuNP-NaCl solution and incubated at room temperature for 4hr.This was followed by the addition of 200 µl of different concentrations of NaCl after every 20 minutes starting from the least to the highest.Confirmation of the association between AuNPs and siRNA was ascertained by gel electrophoresis and staining with safe red dye (18).

Transfection of siRNA -AuNPs complexes into bacterial cells
For transfection of siRNA -AuNPs into bacterial cells, overnight cultures of P. aeruginosa were cultured with siRNA loaded AuNPs, and then incubated at 37֠ C in shaker incubator for 4 hours (18).

RNA extraction and RT-qPCR
In order to determine the expression of MexA gene of different P. aeruginosa isolates, RNA was extracted using RNA extraction kit TransZol Up Plus RNA purification Kit (TransGen,China) following the manufacturer's instructions.The RNA expression of MecA gene was determined by qPCR using the SYBR Green GoTaq® qPCR master mix (Promega, USA) with the recA house-keeping gene used as reference.
The primers used in RT-qPCR for MecA genes are presented in Table 1.The cycling conditions used are as given in Table 2.The gene expression was measured by using the ΔΔCT method (19).

Statistical analysis
Statistical analysis in this study was carried out using the statistical software program SPSS v. 24.0.The MexA gene expression levels were compared using the F test.The Duncan test was used to compare among mean levels of MexA gene and controls.A value P≤0.05 was considered significant.

RESULTS
The study involved the isolation of P. aeruginosa from clinical samples of which four isolates (No. 49, 67, 68, and PDR (5P) were further studied.All of these isolates confirmed the presence of MexA gene by amplifying a 583bp DNA fragment of the corresponding efflux pump by PCR (Fig. 1).Results for the antibacterial effect of gold nanoparticles (AuNPs) against P. aeruginosa isolates, revealed that the various concentrations of AuNPs used to have negative bacterial effect on the bacterial growth which is evidenced by the resazurin dye color change from blue to pink (Fig. 2).

MexA gene silencing and antimicrobial resistance
For studying the effect of MexA gene silencing, AuNPs and siRNA were first conjugated and the conjugation confirmed by gel electrophoresis and staining with safe red dye (Fig. 3).The siRNA loaded onto AuNPs were studied for their impact on MexA gene expression and on the antibiotic resistance in P .aeruginosa.qRT-PCR results showed the siRNA-AuNP complex to significantly reduce the MexA gene expression levels in all isolates tested in comparison to control (Fig. 4).MexA expression fold change ΔΔCT -2 were 0.202, 0.040, 0.063, and 0.163 for P. aeruginosa isolates 67, 66, 49, and PDR (5P) respectively.The statistical analysis for MexA gene expression in this present study showed there are significant differences (P<0.05)among levels of MexA gene within bacterial isolates and controls.The bacterial isolates 66, 49, 5p, and 67 scored lowest levels (0.040 ±0.011, 0.063 ±0.021, 0.163 ±0.032, and 0.202 ±0.092) than controls (1.000 ±0.000), as shown in Table 3. Small letters refer to significant difference (p<0.05) The antibiotic resistance percentage for each clinical isolate of P. aeruginosa to 17 different antibiotics tested was compared before and after siRNA gene silencing of MexA gene (Fig 5).Results showed that the antibiotic resistance percentage altered after the MexA gene was silenced.Before silencing, the isolates' levels of antibiotic resistance varied, ranging from 5.80% to 52.90%.However, the resistance percentages showed considerable variations once the MexA gene was silenced.Notably, the resistance percentage sharply increased, with values varying from 76.40% to 88.20% in P. aeruginosa isolates (No. 66, 49, and 67).However, P. aeruginosa isolate PDR5 (5p) demonstrated total resistance (100%) before MexA gene silencing, which fell to 82% after MexA gene silencing (Fig. 5).The percentage of antibiotic resistance of P. aeruginosa isolates to each of the 17 antibiotics studied before and after silencing of the MecA gene is presented in Table 4.As seen, except for the antibiotics Cefazolin and Ticarcillin/Clavulanic acid, the percentage of antibiotic resistance increased after siRNA silencing of the MecA gene (Table 4).In this study we attempted to investigate the siRNA mediated silencing of the MexA gene, and the MexAB-OprM efflux pump system in P. aeruginosa, and its influence on resistance to different antibiotics.Results showed that siRNA could silence P. aeruginosa MexA gene expression which is in accordance with an earlier study (13).Our study also showed that except for the antibiotics Cefazolin and Ticarcillin/Clavulanic acid, the percentage of antibiotic resistance decreased after siRNA silencing of the MexA gene.This indicates that silencing of the MexA efflux pump gene has a substantial impact on the bacterium's ability to resist antibiotics.Considering that clinical isolates of.P. aeruginosa are multidrug resistant and known to harbor the MexA gene (20,21), the silencing of MexB gene expression could be viewed as a promising mechanism for inducing antibiotic sensitivity.Previous studies have shown that siRNA duplex molecules and siRNAs expressed by plasmid vectors to be effective in gene silencing (10,22).Although metal-based nanoparticles in combination with biomolecules from plants and microorganisms have received attention in medicine (23)(24)(25), gold nanoparticles (AuNPs) are still a popular choice for targeted transport in a variety of biological applications (26).Since, in this investigation we used single-strand siRNA loaded onto AuNPLs for efficient delivery and temporary silencing of the MexA gene of P. aeruginosa, future research should therefore focus on developing a safe and effective siRNA carrier molecule for use in human clinical applications.

CONCLUSION
Pseudomonas aeruginosa's MexA gene expression was suppressed by utilization of single-stranded siRNA loaded onto nanoparticles.The MexA gene suppression was seen to influence antibiotic resistance of the bacterium.

Fig. 2 :
Fig. 2: Resazurin dyes test for effect of gold nanoparticles on P. aeruginosa growth.Pink color indicates no antibacterial effect

Fig. 5 :
Fig. 5: Antibiotic resistance percentage for P. aeruginosa clinical isolates before and after MexA gene silencing

Table 1 :
Primers used in qRT-PCR

Table 2 :
Real time PCR cycling program

Table 3 :
Statistical analysis for MexA gene expression

Table 4 :
Susceptibility to antibiotics before and after silencing of P. aeruginosa MecA gene