Effect of Salicylic Acid on the gene expression of FnbA and FnbB genes in Staphylococcus hominis

Abstract

BACKGROUND:

Staphylococcus hominis is an opportunistic pathogen that expresses surface proteins, which are adhesive proteins that play a major role in biofilm formation. Biofilm is a protective layer that provides S. hominis bacteria with greater antibiotic resistance and promotes its adherence to biomedical surfaces, facilitating its entry into the bloodstream.

OBJECTIVE:

This research aimed to investigate the activity of Salicylic Acid (SA) and its effect on the gene expression of biofilm genes (FnbA and FnbB genes).

METHODS:

A total of 150 blood specimens were collected from patients. The specimens were cultured in broth media of the BacT/ALERT^® system and subcultured on blood and chocolate agar. Bacteria were detected using the VITEK2 system. FnbA and FnbB genes were detected using PCR. The broth microdilution method performed the minimum inhibitory concentration (MIC) of Salicylic acid (SA) on S. hominis isolates with both genes. Detection of the gene expression levels of FnbA and FnbB genes was assessed using Real-Time PCR(RT-PCR).

RESULTS:

The results showed that out of the 150 specimens collected, 35 were S. hominis. The detection of S. hominis bacteria was performed by PCR amplification of two genes FnbA and FnbB and showed 100% and 17.14% of isolates were positive for genes FnbA and FnbB, respectively. The expression of FnbA and FnbB genes was decreased in samples treated with SA compared with untreated ones.

CONCLUSION:

In conclusion, there is a significant impact of SA on the prevention of biofilm formation of S. hominis through the suppression of gene expression, specifically FnbA and FnbB. This could enhance susceptibility to antimicrobial treatments. However, more research is required to determine whether SA leads to the selection of resistant bacteria.

Keywords

Staphylococcus hominis PCR RT-PCR FnbA FnbB salicylic acid

1. Introduction

Staphylococcus hominis is a type of Coagulase-negative staphylococci (CoNS) bacteria of the genus Staphylococcus, consisting of Gram-positive, spherical cells in clusters [1]. It is found on the human skin as normal flora and is usually harmless; however, it can cause infections in people with abnormal and weak immune systems. It produces several proteases, lipases, and exoenzymes that possibly contribute to the persistence of coagulase-negative Staphylococci in the host and may cause tissue degradation. The formation of multilayered bacterial biofilms is a major factor in the pathogenesis of biomaterial-associated infections, which allow microorganisms to adhere to smooth plastic surfaces, colonizing catheters, pacemakers, and various prosthetic and tissue surfaces [2]. Therefore, S. hominis was one of the three most common isolates as nosocomial. Most frequently, these bacteria are recognized as potentially opportunistic pathogens that can infect the gastrointestinal tract, bones, and joints, endocarditis, surgical site infections, and bloodstream infections [3]. Typically, biofilms are microbial colonies that are surface-associated and encased in an extracellular polymeric substance (EPS) matrix. In addition to providing the protection offered by the matrix, bacteria in biofilms employ several survival strategies to evade host defense systems and antibiotics by staying dormant and hiding from them. Within biofilms, bacteria adapt to environmental anoxia and nutrient limitation by altering their metabolism, protein production, and gene expression, leading to decreased metabolism and cell division rates [4, 5]. The production of biofilms occurs in three steps: bacterial cell attachment to the surface, biofilm development, and dispersal of some bacteria from the mature biofilm to new potential infection sites in the host [6]. S. hominis is a common nosocomial that can occasionally cause severe infections [7, 8]. Because of the rise in the use of medical equipment, such as vascular grafts, prosthetic heart valves, invasive surgical procedures, intravenous catheters, and devices for treating joint disease, the coagulase-negative Staphylococcus hominis strain is a common pathogen implicated in bacteremia [9]. Staphylococcai are Gram-positive, facultatively anaerobic, non-motile, and non-spore-forming cocci [10]. This bacterium is positive for catalase and, under a microscope, has a smooth, slightly spherical shape, arranged in grape-like clusters [11]. The pathogenicity of S. hominis is typically recognized by hospital-acquired bacteremia caused by medical interventions [12]. This is due to the virulent ability of these bacteria to produce biofilms. Biofilm formation is the main reason for resistance to the host immune response and antibiotics [13, 14]. The biofilm is frequently associated with infection, which offers a favorable environment for bacterial colonization and can support complex biofilm flora containing many bacteria [15]. Staphylococci express many surface proteins, including fibronectin-binding Proteins A and B (FnBPA and FnBPB), which are adhesives from the MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) family. According to the definition of MSCRAMM, “Has Similarities in structure and function as well as a shared ligand-binding mechanism carried out by two nearby IgG-like folded domains”, and is applied to cell wall-anchored proteins [16]. Other biofilm-forming genes, such as IcaA and IcaB, encode the transmembrane protein, which is involved in the secretion and elongation of the growing polysaccharides of biofilms [17]. Salicylic acid (SA) has general considerations, including antibacterial role (Gram positive and Gram negative), increasing/decreasing efflux pumps, OMPs, upregulating antibiotic targets, and Anti-chlamydial activity at high doses [18]. Recent studies have shown the effect of SA on Staphylococcus species in biofilm formation, the expression of some virulence factors, and the Agr quorum sensing system, a key pathogenicity regulator that is essential to the spread of mature biofilms and aids in their transportation to new colonization sites. SA interferes with agr expression, which limits the dissemination of biofilms [19, 20]. Staphylococcus species adhere to fibronectin, fibrinogen, and elastin of the host by fibronectin-binding proteins A and B (FnBPA and FnBPB), which are encoded by two genes, fnbA and fnbB [21]. SA can form complexes with iron cations and impacts the expression of virulence factors in S. aureus. It has also been demonstrated that the formation of the S. aureus biofilm is inhibited by several iron-chelating molecules. In addition, SA can alter the metabolism and/or metabolic regulators thus changing the gene expression of the essential polysaccharides involved in biofilm formation. In Pseudomonas (Gram-negative bacteria), SA also significantly reduced biofilm production ( $p<$ 0.01) by decreasing the expression of lasI, lasR, rhlI, rhlR, pqsA, and pqsR genes [22]. The aim of this study was to evaluate the gene expression of FnbA and FnbB before and after dealing with Aspirin (Salicylic acid).

2. Materials and methods

2.1 Collection of specimens

The time frame between October 2022 and April 2023 was when the study specimens were collected. There were 150 clinical specimens collected from Bloodstream infections from hospitalized patients with symptoms and signs of bacteremia. Some patients are asymptomatic or have only mild fever, whereas some symptoms increase, such as shaking chills, tachypnea, hypotension, persistent fever, altered sensorium, and gastrointestinal symptoms (nausea, abdominal pain, vomiting, diarrhea) [23]. The patients ranged in age from newborns (3 days) to adults (up to 88 years) who were admitted to hospitals in Baghdad. Blood samples from distinct vein punctures are collected in the hospital’s aseptic laboratory. To the greatest extent feasible, blood was drawn simultaneously from two arms, greater or equal to 10 ml blood for adults and 1–5 ml for infants, using a sterile syringe and needle by venipuncture. The needle was changed and immediately transferred into BacT/ALERT^® Plus Blood Culture Bottles [24].

Table 1
The primers used in this research

No.	Genes	Sequences	The product size	Annealing temperature	Sources
1	16S rRNA	F: TGTCGTGAGATGTTGGG R: CGATTCCAGCTTCATGT	270 bp	52	[40]
2	FnbA	F: CCCTCTTCGTTATTCAGCC R: CAGGAGGCAAGTCACCTTG	422 bp	59	[31]
3	FnbB	F: TAAACACCGACGATAATAACCAAA R: GGTCTAGCCTTATTTTCATATTCA	495 bp	60	[41]

2.2 Isolation and identification

2.2.1 Culture

After injecting the blood patient into culture bottles (broth media), it was incubated in a BacT/ALERT^® system (BioMe’rieux, USA) at 37^∘C for 5 days from the culture bottles, which were marked as a positive alarm in this incubation instrument. Subcultures were performed, and the specimen was streaked both aerobically and anaerobically on chocolate and blood agar media. For a full day, the plates were incubated at 37^∘C. Gram staining, biochemical testing, and morphological characteristics of the subcultured colony were performed (Gram-positive coccus is found as pairs, less frequently as irregular, grape-like clusters tetrads, cells are coagulase-negative, catalase positive, exhibit facultatively anaerobic metabolism and able to use glucose oxidatively and fermentatively). The Vitek2 compact system was utilized, and molecular detection was used to verify the identity [25, 26].

2.2.2 Molecular detection of FnbA and FnbB genes

The sequences and sizes of amplicons for virulence genes FnbA and FnbB were verified using the BLAST program from the NCBI database, and the diagnosis was performed using conventional PCR and analyzed by gel electrophoresis techniques to identify the right size of the amplified DNA bands.

Primers

All primers were provided by Macrogen Company in lyophilized form, as shown in Table 1.

DNA extraction

DNA was extracted from the isolated S. homoinis using a purification method called the Genomic DNA Mini Kit (Geneaid, Taiwan), and the DNA was extracted using the bacterial protocol for Gram-positive. The genomic DNA extraction from S. hominis isolates was confirmed as bands by gel electrophoresis. The Nanodrop spectrophotometer was used to measure the concentration and purity of DNA. The DNA purity and integrity were determined by the ratio of absorbance at 260 and 280 nm. It is generally agreed upon that DNA is “pure” at a ratio of approximately (1.8). A significantly lower ratio ( $\leqslant$ 1.6) could suggest the presence of proteins, phenol, or other pollutants that are strongly absorbed at or near 280 nm at near 280 nm [27]. The PCR reaction output was confirmed using 1.5% (wt/vol) agarose gel electrophoresis and ultraviolet light. The gel was run for 60 min at 100 V using a 100-bp DNA ladder (Promega, USA). Using a Gel imaging system, PCR products were visualized after being stained with ethidium bromide [28].

PCR master mix

Using 25 $\mu$ L of PCR reaction mixtures as follows: (12.5 $\mu$ l Tag Green Master Mix, 1 $\mu$ l Forward Primer (10 pmol/ $\mu$ L), 1 $\mu$ l Reverse Primer (10 pmol/ $\mu$ L), 5.5 $\mu$ l Nuclease Free Water, and 5 $\mu$ l Extracted DNA.

PCR program

Table 2
The PCR program to identify the FnbA gene of S.hominis isolates

No.	Steps	Temperature (^∘C)	Times	No. of cycles
1	Initial denaturation	Ninety-five	5 minute.	1
2	Denaturation	Ninety-five	30 second.
3	Annealing	Fifty-nine	30 second.	30
4	Extension	Seventy-two	30 second.
5	Final extension	Seventy-two	7 minute.	1

Table 3

The PCR program to identify the FnbB gene of S.hominis isolates

No.	Steps	Temperature (^∘C)	Times	No. of cycles
1	Initial denaturation	Ninety-five	5 minute.	1
2	Denaturation	Ninety-five	30 second.
3	Annealing	Sixty	30 second.	30
4	Extension	Seventy-two	30 second.
5	Final extension	Seventy-two	7 minute.	1

RCR is technology used for amplifying DNA sequences, which is based on the principle of enzymatic replication of the nucleic acids. To detect the FnbA and FnbB genes of S. homoinis, PCR was performed in a thermal cycler (Applied Biosystem, 9902, Singapore) in accordance with the PCR program with some modifications. Optimization of PCR was accomplished after several trials (Tables 2, 3).

PCR product analysis

The analysis of PCR products of the FnbA, FnbB gene of S. hominis were performed on 1.5% agarose gel. PCR products were loaded directly. For the PCR product, 5 $\mu$ l was directly loaded onto wells and 5 $\mu$ l deionized water was loaded in one well, which was used as the negative. The gel was run at 100 volts for 60 minutes at room temperature using a 100-bp DNA ladder (Promega, USA). The singleplex PCR products were observed using an image analyzer (Chemi Imager 5500, Alpha Innotech, USA) after staining with ethidium bromide [28].

2.3 Minimum inhibitory concentrations (MIC) of Aspirin (Salicylic acid)

Minimum inhibitory concentrations (MICs) are defined as the lowest concentration of antimicrobial agents that inhibit the growth of a microorganism after a period of incubation. The minimum inhibitory concentrations (MICs) of the products (Salicylic acid solutions) were measured using a Resazurin dye. MIC was determined for the six samples that contained both genes (FnbA, FnbB). The MIC was calculated using the microdilution technique (Microtiter Plate Assay) with Resazurin Dye (method using the oxidation-reduction colorimetric indicator resazurin), at doses ranging 1000 ug/ml [29, 30]. The bacterial inoculum for the test was produced by culturing a Staphylococcus hominis subculture in brain heart agar for 18–24 h at 37^∘C. Wells without Aspirin were used as positive controls, whereas those without S. hominis were considered as negative controls. Following a 24-h incubation period at 37^∘C, the wells were examined visually to check for growth. According to some studies, the minimum inhibitory concentration (MIC) of aspirin inhibits bacterial growth [30].

2.3.1 Quantitative MIC of Aspirin (Salicylic acid) [31]

1.
Using the micropipette, Put 100 ul of the medium into all the wells. of a microtiter plate.
2.
Pipette 100ul of appropriate 1000 ug/ml of Salicylic acid solutions (prepared by adding 1000 ug/ml of Salicylic acid in 1 ml of 0.2 M dimethyl sulfoxide) through the wells within column 1 except raw A, which is used as a positive control.
3.
Utilize the micropipette set at 100 ul, and mix the product through column 1’s wells by sucking up and down from six to eight times. Avoid splashing.
4.
Take 100 ul out of column 1 and put it in column 2, Column 2 thus represents a twofold dilution of column 1, Transfer 100 ul from column 2 to column 3, Proceed as before, stopping at column 11.
5.
Throw away 100 ul from column 11 instead of putting it in column 12.
6.
With the smaller micropipette set to 10 ul, Add bacteria to every well except raw H (sterility control and blank for the plate scanner).
7.
The plates should be incubated at 37^∘C temperature.
8.
After 24 hours of satisfactory growth, 20 $\mu$ l of resazurin was added to each well, and the mixture was incubated for a further 2–4 hours at 37^∘C. A pink color shift was observed and noted. The minimum inhibitory concentration was determined to be the concentration at which the color change occurred (MIC).

Table 4
RT-PCR component

No. of reaction 12 Rxn No. of primers 6

Reaction Volume /run 10 $\mu$ l No. of PCR cycles 40

Table 5
RT-PCR master mix

Master mix component Stock Final concentration Volume

qPCR Master Mix 2X 1X 5

Forward primer 10 $\mu$ M 1 $\mu$ M 0.5

Reverse primer 10 $\mu$ M 1 $\mu$ M 0.5

Nuclease free water 2.5

RT mix 50X 1X 0.25

Mgcl2 0.25

RNA 1

Total volume 10

Table 6
The real-time PCR program

Steps Temperatures Times No. of cycles

RT. Enzyme Activation 37^∘C 15 min. 1

Initial denaturation 95^∘C 5 min.

Denaturation 95^∘C 20 sec. 40

Annealing 52 or 59 or 60^∘C 20 sec.

Extension 72^∘C 20 sec.

2.4 Gene expression

Master mix component	Stock	Final concentration	Volume
qPCR Master Mix	2X	1X	5
Forward primer	10 $\mu$ M	1 $\mu$ M	0.5
Reverse primer	10 $\mu$ M	1 $\mu$ M	0.5
Nuclease free water			2.5
RT mix	50X	1X	0.25
Mgcl2			0.25
RNA			1
Total volume			10

Steps	Temperatures	Times	No. of cycles
RT. Enzyme Activation	37^∘C	15 min.	1
Initial denaturation	95^∘C	5 min.
Denaturation	95^∘C	20 sec.	40
Annealing	52 or 59 or 60^∘C	20 sec.
Extension	72^∘C	20 sec.

Gene expression of FnbA, FnbB, and $16S$ (housekeeping) genes was evaluated via using the Rotor-Gene Q 6 plex platform (Qiagen, USA) for all study groups. The fluorescent dye SYBR green was used to identify double-stranded DNA, including cDNA [32]. The main goal was to compare the expression of these genes with and without salicylic acid, to better understand the role of salicylic acid on these genes in S. hominis resistance. The cycle threshold (Ct) values were obtained from the rotor gene for both the target and reference genes, the values above 35 were regarded as non-specific and excluded from the study. The obtained Ct values were normalized to the Ct value of the reference gene as an internal control relation ( $\Delta$ Ct $=$ Ct gene of interest – Ct reference gene) for each sample and normalized to the control sample (untreated), ( $\Delta\Delta$ Ct $=$ $\Delta$ Ct of treated samples with salicylic acid – $\Delta$ Ct of untreated samples with salicylic acid). Then, the 2 ( $-\Delta\Delta$ Ct) value was calculated [17]. The results were expressed as relative expression of FnbA and FnbB genes to 16SrRNA gene. The experiment used six clinical isolates of S. homoinis that were positive for genes FnbA and FnbB, then incubated with sub-MICs of salicylic acid. Expression of these genes in the isolates was measured before and after treatment with salicylic acid. The different concentrations of Salicylic acid (3.9–1.95) $\mu$ g/ml used in the treatment were below the MIC values and were applied to the bacterial isolates. The tubes were incubated for 24 h at 37^∘C [33]. The Promega kit was used in this experiment, and the reaction setup and thermal cycling protocol (one-step RT-PCR (Tables 4, 5, 6).

Statistical analysis was used to determine how various factors affect the study’s parameters using the Statistical Analysis System- SAS (2018). To make a significant comparison between means, the T-test was used. In this study, the chi-square test was used to compare the percentages (0.05 and 0.01 probability) [34].

2.4.1 RNA Extraction for S. hominis.

The RNA extraction was done by using TRIzol^® Reagent (Cat. No. 15596026) provided by Thermo Scientific Company (USA), chloroform (Cat. No. 1024441000) provided by LiChrosolv (Germany) [35]. The concentration and purity of RNA were measured by Quantus fluorometer (Promega, USA), then converted to cDNA for analysis and calculation of the expression levels. The procedures include data analysis, qPCR amplification, and total RNA purification. The figure below illustrates the study’s experimental design and workflow (Fig. 1).

Figure 1.

Schematic outlining the overall experimental design.

Figure 2.

The Gel electrophoresis of PCR products of FnbA gene. Detection of FnbA gene (422 base pair) by gel electrophoresis technique, the gel was run at 100 volt for 60 minutes at room temperature using a 100-bp DNA ladder (M), the negative control (NC) 5 $\mu$ l deionized water was loaded. The number of $S$ . hominis isolates detected to have this gene was 35 samples (100%). The singleplex PCR products were observed using an image analyzer after staining with ethidium bromide.

Figure 3.

The Gel electrophoresis of PCR products of FnbB gene. Detection of FnbB gene (495 base pair) by gel electrophoresis technique, the gel was run at 100 volt for 60 minutes at room temperature using a 100-bp DNA ladder (M), the negative control (NC) 5 $\mu$ l deionized water was loaded. The number of $S$ . hominis isolates detected to have this gene was only 6 samples (17.14%). The singleplex PCR products were observed using an image analyzer after staining with ethidium bromide.

3. Results and discussion

The results demonstrate that Staphylococcus hominis is one of the common bacteria isolated from patients’ blood in hospitals in Baghdad city. S. hominis treated with a subMIC of salicylic acid solution had lower gene expression for adhesion-encoding genes (FnbA and FnbB genes) than those untreated.

3.1 Isolation and identification

A total of 150 clinical specimens isolated from blood were collected and cultured. The results included 35 (28.45%) specimens of blood that were subjected to typical biochemical tests and characteristics of morphology that are specific to coagulase-negative Staphylococcus hominis, whereas the other 88 (71.55%) isolates belong to other species of staphylococci and other pathogenic bacteria. while 27 clinical specimens showed no growth. In this study, 67 (54.47%) patients were females, whereas 56 (45.52%) were men. Patients’ ages for the study were between newborns (3 days) and adults (up to 88 years) for both genders. In a similar study, most pathogens isolated from blood cultures were Coagulase-negative Staphylococcus bacteria 60 (30.77%) [36], While in another study Coagulase-negative Staphylococcus bacteria in blood culture were 16 (10.09%) [37].

Table 7
Fold change of FnbA gene expression in Staphylococcus hominis treated and untreated with salicylic acid

Means having with the different letters in same column differed significantly, ^** ( $P\leqslant$ 0.01).
Sample	16SrRNA ct	FnbA ct	$\Delta$ CT	$\Delta\Delta$ CT	Folding
Control	12.89	16.88	3.995	0.00	1.00 $\pm$ 0.00 d
7	15.24	15.26	0.02	$-$ 4.03	16.39 $\pm$ 1.37 a
20	9.86	16.89	7.03	1.91	0.27 $\pm$ 0.03 d
35	7.62	15.60	7.98	1.06	0.48 $\pm$ 0.07 d
11	13.64	13.65	0.01	$-$ 1.56	2.94 $\pm$ 0.22 c
16	12.28	16.15	3.87	0.36	0.78 $\pm$ 0.16 d
33	13.64	14.32	0.69	$-$ 2.11	4.33 $\pm$ 0.57 b
LSD ( $P$ -value)	—	—	—	—	1.439^** (0.0001)

The fold change compared to the control group (untreated), $P\leqslant$ 0.01 is significant (s), and $P>$ 0.01 is non-significant (NS).

Table 8

Fold change of FnbB gene expression in Staphylococcus hominis treated and untreated with salicylic acid

Means having with the different letters in same column differed significantly, ^** ( $P\leqslant$ 0.01).
Sample	16SrRNA	FnbB	$\Delta$ CT	$\Delta\Delta$ CT	Folding
Control	12.89	29.88	16.99	0.00	1.00 $\pm$ 0.00 c
7	15.24	28.86	13.62	$-$ 2.58	6.00 $\pm$ 0.52 b
20	9.86	29.87	20.01	0.85	0.55 $\pm$ 0.02 c
35	7.62	31.09	23.47	3.12	0.11 $\pm$ 0.02 c
11	13.64	24.97	11.33	$-$ 4.63	24.71 $\pm$ 3.92 a
16	12.28	30.03	17.76	3.16	0.11 $\pm$ 0.02 c
33	13.64	29.79	16.15	0.46	0.73 $\pm$ 0.05 c
LSD ( $P$ -value)	—	—	—	—	1.297 ^** (0.0001)

The fold change compared to the control group (untreated), $P\leqslant$ 0.01 is significant (s), and $P\leqslant$ 0.01 is non-significant (NS).

Figure 4.

MICs of Aspirin (Salicylic acid) by ug/ml. MIC and sub-MIC determine of SA solution, serially diluted twofold from (500–0.48 $\mu$ g/ml). After an overnight incubation followed by a 2–4 hours incubation with resazurin, all negative control (NC) wells remained blue, all the wells in the positive control (PC) wells (which included both growing bacteria and medium) of all tested microorganisms transformed from blue to pink.

Figure 5.

Gene Expression of FnbA gene of S. hominis with and without salicylic acid using RT-PCR technique. CT cycle RT-PCR of FnbA gene befor and after treated with Salicylic Acid.

Figure 6.

Gene Expression of FnbB gene of S. hominis with and without salicylic acid using RT-PCR technique. CT cycle RT-PCR of FnbB gene befor and after treated with Salicylic Acid.

3.2 Molecular identification of FnbA and FnbB gene in S. hominis

DNA was amplified for these genes using PCR in a mono-plex pattern using specific primers. The amplification revealed a product of 422 bp (FnbA gene) and 495 bp (FnbB gene). The number of $S$ . hominis isolates that have the FnbA gene was 35 samples (100%), and the number of S. hominis isolates that have the FnbB gene was only 6 samples (17.14%), Figs 2 and 3. In another study, the same adhesion-encoding genes were detected in S. aureus. The fnbA gene was detected in the isolates 32 (100%), whereas fnbB was 8 (25%) [38].

3.3 The minimum inhibitory concentration (MICs) of Salicylic acid solution

The products were serially diluted twofold from (500 $\mu$ g/ml to 0.48 $\mu$ g/ml). All sterility control wells remained blue after an overnight incubation followed by a 2–4 hours incubation with resazurin. In compression, all wells in the positive control column (which included both growing bacteria and medium) of all tested microorganisms transformed from blue to pink or pale pink, as shown in (Fig. 4). MIC of Salicylic acid for investigated isolates was a ranged from (7.81–3.9 ug/ml).

3.4 Gene Expression using the RT-PCR technique

For the FnbA gene, when using the sub-MIC values of salicylic acid with the isolates, it was found that the fold of gene expression was slightly elevated before treatment with salicylic acid, whereas the fold change of the isolates after treatment with salicylic acid was lower (0.27–0.48–0.78) (Table 7), (Fig. 5). The calculation of fold values was performed using the average of triplicates of the expression of each gene.

For the FnbB gene, when using the sub-MIC values of salicylic acid with the isolates, it was found that the fold of gene expression was slightly elevated before the treatment with salicylic acid, whereas the fold change of the isolates after treatment with salicylic acid was lower (0.55–0.11–0.11–0.73) (Table 8), (Fig. 6).

We concluded that the low gene expression of FnbA and FnbB genes of S. homoinis isolates. Salicylic acid showed an antibacterial effect and could be important for the advancement of antibacterial agents against S. homoinis, particularly in multidrug-resistant isolates. In [15], SA led to decreased expression of some fibronectin and fibrinogen-binding protein genes in Staphylococcus aureus.

The integrity of RNA during storage is one of the crucial issues that needs to be considered. and the gene expression procedure. Because RNA can be affected by degradation reactions, it is sensitive to oxidation outside the cell by mechanisms generally involving metallic ions, which also result from an atmospheric pollutant that rapidly reacts with RNA. To prevent degradation, RNA samples are generally stored frozen at $-$ 20^∘C or $-$ 80^∘C or under liquid nitrogen in sterile conditions [39].

4. Conclusion

Molecular detection using the PCR technique showed (100%) of isolates possess the FnbA gene and (17.14%) FnbB gene. The findings showed that the gene expression levels of biofilm formation genes were lower in strains treated with sub-MICs of salicylic acid than in untreated strains. This decline in the expression of biofilm formation genes corresponds to previous results demonstrating that sub-MICs of Salicylic acid inhibit biofilm formation and bacterial growth. There was a significant difference in the expression of FnbA and FnbB genes in the SA-treated isolates compared with the untreated ^** ( $P\leqslant$ 0.01).

Footnotes

Acknowledgments

The authors gratefully acknowledge all the staff in the Institute of Genetic Engineering and Biotechnology/University of Baghdad and the Department of Bacteriology Laboratory in Medical City Hospitals, Baghdad, Iraq.

Conflict of interest

The authors affirm that they have no competing interests.

Author contributions

HAA and ZHA Conceptualisation; Interpretation or analysis of data; HAA: Formal analysis and investigation and Resources; ZHA: funding acquisition and supervision; HAA: Writing original draft preparation; ZHA: writing review and editing.

References

Muhammad

and Al-Mathkhury

, The Prevalence of methicillin resistant Staphylococcus aureus and methicillin resistant Staphylococcus epidermidis in AL-Sulaimania city, Iraqi Journal of Science 55 (2014), 386–393.

Malik

, Isolation, Speciation, Detection of Virulence Factors and Antimicrobial Susceptibility Pattern in Clinical Isolates of Coagulase-Negative Staphylococci at a Tertiary Care Hospital, in, Rajiv Gandhi University of Health Sciences (India), 2018.

Noshak

M.A.

Rezaee

M.A.

Hasani

and Mirzaii

, The role of the coagulase-negative staphylococci (CoNS) in infective endocarditis; a narrative review from 2000 to 2020, Current Pharmaceutical Biotechnology 21 (2020), 1140–1153.

Hall-Stoodley

and Stoodley

, Evolving concepts in biofilm infections, Cell Microbiol 11(7) (2009 Jul), 1034–43, in, DOI.

Donlan

R.M.

and Costerton

J.W.

, Biofilms: survival mechanisms of clinically relevant microorganisms, Clinical Microbiology Reviews 15 (2002), 167–193.

Otto

, Staphylococcal infections: mechanisms of biofilm maturation and detachment as critical determinants of pathogenicity, Annual Review of Medicine 64 (2013), 175–188.

Berends

M.S.

Luz

C.F.

Ott

Andriesse

G.I.

Becker

Glasner

and Friedrich

A.W.

, Trends in occurrence and phenotypic resistance of coagulase-negative Staphylococci (CoNS) found in human blood in the Northern Netherlands between 2013 and 2019, Microorganisms 10 (2022), 1801.

Dahash

S.L.

Dawood

S.D.

and Mahmoud

I.S.

, Staphylococcal infections among leukemic patients, Baghdad Science Journal 9 (2012), 270–276.

Asante

Amoako

D.G.

Abia

A.L.

Somboro

A.M.

Govinden

Bester

L.A.

and Essack

S.Y.

, Review of clinically and epidemiologically relevant coagulase-negative staphylococci in Africa, Microbial Drug Resistance 26 (2020), 951–970.

10.

Mohammed

S.W.

and Radif

H.M.

, Detection of icaA gene expression in clinical biofilm-producing Staphylococcus aureus isolates, Iraqi Journal of Science (2020), 3154–3163.

11.

Kadhum

H.H.

and Abood

Z.H.

, Staphylococcus aureus Incidence in Some Patients with a Topic Dermatitis in Baghdad City, Iraqi J. Biotech 21 (2022), 13–20.

12.

Szemraj

Grazul

Balcerczak

and Szewczyk

E.M.

, Staphylococcal species less frequently isolated from human clinical specimens-are they a threat for hospital patients? BMC Infectious Diseases 20 (2020), 1–10.

13.

Jaddoa

and Al-Mathkhury

H.J.F.

, Biofilm Shows Independency form Hemolysin Genes Arsenal in Methicillin Resistant Staphylococcus aureus, Iraqi Journal of Science 59 (2018), 2184–2194.

14.

Mohaisen

S.H.

Ali

M.H.

Shehab

Z.H.

Al-Mayyahi

and Abdulhassan

, Effect of ultraviolet light on the expression of icaD gene in Staphylococcus aureus local isolates in Iraq, (2021).

15.

Ahmed

S.T.

Abdallah

N.M.

AL-Shimmary

S.M.

and Almohaidi

A.M.

, The role of genetic variation for icaA gene Staphylococcus aureus in producing biofilm, Hospital 3 (2021), 4.

16.

Foster

T.J.

Geoghegan

J.A.

Ganesh

V.K.

and Höök

, Adhesion, invasion and evasion: the many functions of the surface proteins of Staphylococcus aureus, Nature Reviews Microbiology 12 (2014), 49–62.

17.

Abdrabaa

M.K.

and Abd Aburesha

, Gene Expression Evaluation of Intracellular Adhesins and Regulatory Genes among Biofilm Producing MRSA Isolates, Iraqi Journal of Science (2023), 75–83.

18.

Di Bella

Luzzati

Principe

Zerbato

Meroni

Giuffrè

Crocè

L.S.

Merlo

Perotto

and Dolso

, Aspirin and infection: A narrative review, Biomedicines 10 (2022), 263.

19.

Dotto

Lombarte Serrat

Ledesma

Vay

Ehling-Schulz

Sordelli

D.O.

Grunert

and Buzzola

, Salicylic acid stabilizes Staphylococcus aureus biofilm by impairing the agr quorum-sensing system, Scientific Reports 11 (2021), 2953.

20.

Sykes

E.M.

White

McLaughlin

and Kumar

, Salicylic acids and pathogenic bacteria: new perspectives on an old compound, Canadian Journal of Microbiology 70 (2023), 1–14.

21.

Saleh

M.M.

Yousef

Shafik

S.M.

and Abbas

H.A.

, Attenuating the virulence of the resistant superbug Staphylococcus aureus bacteria isolated from neonatal sepsis by ascorbic acid, dexamethasone, and sodium bicarbonate, BMC Microbiology 22 (2022), 268.

22.

Arriero

M.M.

de La Pinta

J.C.

Escribano

Celdrán

Muñoz-Alameda

García-Cañete

Jiménez

A.M.

Casado

Farré

and López-Farré

, Aspirin Prevents Escherichia coli Lipopolysaccharide – and Staphylococcus aureus – Induced Downregulation of Endothelial Nitric Oxide Synthase Expression in Guinea Pig Pericardial Tissue, Circulation Research 90 (2002), 719–727.

23.

Andersson

Axelsson

Larsson

Bremer

Gellerstedt

Bång

Herlitz

and Ljungström

, The early chain of care in bacteraemia patients: early suspicion, treatment and survival in prehospital emergency care, The American Journal of Emergency Medicine 36 (2018), 2211–2218.

24.

Dilnessa

Demeke

Mengistu

and Bitew

, Emerging blood culture technologies for isolation of blood pathogens at clinical microbiology laboratories, J Med Microb Diagn 5 (2016), 1–7.

25.

Abdul-Rahman

S.M.

and Khider

A.K.

, Neonatal sepsis: Bacteriological profile, molecular detection and antimicrobial susceptibility test among pre-term pediatrics in Erbil city, Iraq, Zanco Journal of Medical Sciences (Zanco J Med Sci) 24 (2020), 256–273.

26.

AlSaadi

B.Q.H.

Mohaisen

KAZAAL

and Abdulla

Z.N.

, Study the prevalence of mecA gene in methicillin resistance staphylococcus aureus (MRSA) isolated from different clinical specimens and their antibiotic resistance profile, Annals of Tropical Medicine and Health 23 (2020), 231–223.

27.

Lucena-Aguilar

Sánchez-López

A.M.

Barberán-Aceituno

Carrillo-Avila

J.A.

López-Guerrero

J.A.

and Aguilar-Quesada

, DNA source selection for downstream applications based on DNA quality indicators analysis, Biopreservation and Biobanking 14 (2016), 264–270.

28.

Sambrook

Fritsch

E.F.

and Maniatis

, Molecular cloning: a laboratory manual, Cold spring harbor laboratory press, 1989.

29.

Elshikh

Ahmed

Funston

Dunlop

McGaw

Marchant

and Banat

I.M.

, Resazurin-based 96-well plate microdilution method for the determination of minimum inhibitory concentration of biosurfactants, Biotechnology Letters 38 (2016), 1015–1019.

30.

Raheem

N.N.

and Ghaima

K.K.

, Antibiofilm Activity of Nystatin, Aspirin and EDTA Against Candida albicans Isolated from Iraqi Women with Vulvovaginitis, Medico-legal Update, 21 (2021).

31.

Kitti

Seng

Thummeepak

Boonlao

Jindayok

and Sitthisak

, Biofilm formation of methicillin-resistant coagulase-negative Staphylococci isolated from clinical samples in Northern Thailand, Journal of Global Infectious Diseases 11 (2019), 112–117.

32.

Rebouças

E.d.L.

Costa

J.J.d.N.

Passos

M.J.

Passos

J.R.d.S.

Hurk

R.v.d.

and Silva

J.R.V.

, Real time PCR and importance of housekeepings genes for normalization and quantification of mRNA expression in different tissues, Brazilian Archives of Biology and Technology 56 (2013), 143–154.

33.

Zhou

J.-W.

P.-C.

Jiang

Tan

X.-J.

and Jia

A.-Q.

, Quorum sensing inhibition and metabolic intervention of 4-hydroxycinnamic acid against Agrobacterium tumefaciens, Frontiers in Microbiology 13 (2022), 830632.

34.

Cary

, Statistical analysis system, User’s guide. Statistical. Version 9, SAS. Inst. Inc. USA, (2012).

35.

Zhao

Luo

Xue

Wang

Han

Heilbronner

and Zhao

, Antimicrobial and anti-biofilm activity of a thiazolidinone derivative against Staphylococcus aureus in vitro and in vivo , Microbiology Spectrum (2024), e02327–02323.

36.

Mahmoudi

Ghasemi Bassir

H.R.

Hosseini

S.M.

Arabestani

M.R.

and Alikhani

M.Y.

, The frequency of bacteria isolated from blood cultures and antibiotic susceptibility patterns among admitted patients in Hospital of Hamedan University of Medical Sciences, Iranian Journal of Medical Microbiology 10 (2016), 69–74.

37.

Opota

Croxatto

Prod’Hom

and Greub

, Blood culture-based diagnosis of bacteraemia: state of the art, Clinical Microbiology and Infection 21 (2015), 313–322.

38.

Al Ani

A.T.A.

and Al Meani

S.A.L.

, Molecular Screening of Adhesion Proteins Genes in Staphylococcus aureus Strains Isolated from Different Clinical Infections in Baghdad City and Identification of Their Relationship with Some Virulence Factors, Al-Nahrain Journal of Science 21 (2018), 79–89.

39.

Fabre

A.-L.

Colotte

Luis

Tuffet

and Bonnet

, An efficient method for long-term room temperature storage of RNA, European Journal of Human Genetics 22 (2014), 379–385.

40.

Stutz

Stephan

and Tasara

, SpA, ClfA, and FnbA genetic variations lead to Staphaurex test-negative phenotypes in bovine mastitis Staphylococcus aureus isolates, Journal of Clinical Microbiology 49 (2011), 638–646.

41.

Arciola

C.R.

Campoccia

Gamberini

Donati

M.E.

and Montanaro

, Presence of fibrinogen-binding adhesin gene in Staphylococcus epidermidis isolates from central venous catheters-associated and orthopaedic implant-associated infections, Biomaterials 25 (2004), 4825–4829.

No. of reaction	12	Rxn	No. of primers	6
Reaction Volume /run	10	$\mu$ l	No. of PCR cycles	40

Effect of Salicylic Acid on the gene expression of FnbA and FnbB genes in Staphylococcus hominis

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Materials and methods

2.1 Collection of specimens

Table 1 The primers used in this research

2.2.1 Culture

2.2.2 Molecular detection of FnbA and FnbB genes

Table 2 The PCR program to identify the FnbA gene of S.hominis isolates

2.3.1 Quantitative MIC of Aspirin (Salicylic acid) [31]

2.4.1 RNA Extraction for S. hominis.

3.1 Isolation and identification

Table 7 Fold change of FnbA gene expression in Staphylococcus hominis treated and untreated with salicylic acid

3.3 The minimum inhibitory concentration (MICs) of Salicylic acid solution

3.4 Gene Expression using the RT-PCR technique

4. Conclusion

Footnotes

Acknowledgments

Conflict of interest

Author contributions

References

Table 1
The primers used in this research

Table 2
The PCR program to identify the FnbA gene of S.hominis isolates

Table 7
Fold change of FnbA gene expression in Staphylococcus hominis treated and untreated with salicylic acid