Potentiating activity of rhein in targeting of resistance genes in methicillin-resistant Staphylococcus aureus

Ryong Gong, Dae Young Lee, Jae Won Lee, Doo Jin Choi, Geum-Soog Kim, Sang Hyuk Lee,Young-Seob Lee

Department of Herbal Crop Research, National Institute of Horticultural & Herbal Science, Rural Development Administration, 92 Bisan-ro, Eumsung,Chungbuk, 27709, Korea

Keywords：Antimicrobials Mechanism of action Staphylococcus aureus Polymerase chain reaction

ABSTRACT Objective: To investigate the synergistic effect between rhein (RHE) and oxacillin against Staphylococcus aureus (MRSA) at the gene level. Method： A minimum inhibitory concentration and checkerboard dilution test were conducted to evaluate antibacterial activity. Reverse transcriptase polymerase chain reaction was conducted to investigate the gene expressions.Results： RHE exhibited a minimum inhibitory concentration of 62.5-250.0 μg/mL against various MRSA strains and the reference strain, respectively. As revealed by the checkerboard assay, a combination of RHE and oxacillin exhibited synergistic or partially synergistic effects against MRSA strains. RHE decreased the expressions of mecA/blaZ in a dose-dependent manner. RHE also decreased the expressions of the regulator genes mecI/blaI and mecR1/blaR1.Conclusions： We suggest that RHE affects the activity of mecR1/blaR1, which is located in the cell membrane of MRSA and results in the suppression of mecA/mecI/mecR1 and blaZ/blaI/blaR1 gene expressions.

1. Introduction

Methicillin-resistantStaphylococcus aureus(S. aureus) (MRSA)causes infections ranging from skin and soft tissue infection to more severe diseases, including bacteremia, osteomyelitis, arthritis,pneumonia, sepsis, and pericarditis[1]. β-Lactams, which are antibiotics that disrupt cell wall synthesis by targeting the fourS.aureuspenicillin-binding proteins involved in the transpeptidation of peptidoglycan, were commonly used to treat bacterial infection[2].However, MRSA has recently developed two major resistance mechanisms against β-lactam antibiotics. First, bacteria produce β-lactamases, a family of enzymes that inactivate β-lactam antibiotics, such as penicillins, carbapenems, and monobactams by hydrolyzing their β-lactam rings[3]. Second, the production of penicillin-binding protein 2A (PBP2a) is a major determinant of β-lactam resistance. PBP2a has a low affinity for β-lactams, thereby allowing it to maintain its transpeptidation activity in the presence of β-lactam antibiotics[4]. β-lactamase and PBP2a are encoded byblaZandmecA, respectively; these are located in a mobile genetic element known as the Staphylococcal cassette chromosome mec(SCCmec). The transcription ofmecAandblaZis regulated by themecI-mecR1andblaI-blaR1systems, respectively. The mec and bla operons are controlled by two-component systems comprising the repressorsmecIandblaIand the sensorsmecR1andblaR1. The genes formecA/blaZ, its repressorsmecI/blaI, and the transducersensorsmecR1/blaR1are clumped together, either on a plasmid or within the bacterial chromosome. In the absence of β-lactam,the DNA repressor MecI/BlaI suppressesmecA/blaZby binding to the conserved DNA motif TACA/TGTA, which is located in the promoter region ofmecA/blaZ. However, when exposed to β-lactam antibiotics, acylation ofBlaR1/MecR1, which is a transmembrane and signal transduction protein, is subsequently followed by autoproteolytic cleavage on the cytoplasmic side of the cell membrane. The cleaved intracellular fragment ofMecR1/BlaR1then moves to the bacterial chromosome where it removes its cognate repressor MecI/BlaI by proteolysis. Transcription of themecA/mecR1/mecIandblaZ/blaR1/blaIgenes begins once the repressor dissociates from the promoter region[5-7].

To overcome β-lactam resistance, antibiotics with different modesof-action or agents that inhibit drug resistance should be used concurrently. Therefore, antibacterial agent combination therapy appears useful because it can enhance antibacterial activity and reduce drug resistance[8]. Rhein (RHE) is a lipophilic anthraquinone compound found in rhubarb (Rheum palmatumL.),Reynoutria japonica(Houtt.), andFallopia multiflora, and is extensively used in treating various clinical disorders, such as hepatic disease,osteoarthritis, atherosclerosis, various cancers, and diabetic nephropathy (Figure 1). RHE was identi fied as a major metabolite of diacerein, a prodrug used in treating osteoarthritis[9-12]. In addition, RHE has been reported to possess antibacterial activity and synergistic effects against MRSA when combined with antibiotics such as ampicillin (AM) or oxacillin (OX)[13].

However, there is no reports which state that RHE inhibits the drug resistance mechanism of MRSA at the molecular level. Here, we investigated the synergistic effect of RHE combined with antibiotics on MRSA drug resistance at the gene level.

Figure 1. Chemical structure of rhein.

2. Materials and methods

2.1. Chemicals

RHE (4,5-dihydroxyanthraquinone-2-carboxylic acid), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),dimethyl sulfoxide (DMSO), AM, and OX were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mueller-Hinton (MH) broth and MH agar were purchased from Difco Laboratories (Baltimore,MD, USA).

2.2. Bacterial strains and growth conditions

Strains 25923 and 33591 were purchased from the American Type Culture Collection (ATCC). Clinical isolates of bacterial strains (DHCR 1-15) were obtained from the Department of Plastic Surgery of Wonkwang University Hospital (Iksan, Korea). Before experimentation, all bacteria were stored in 30% glycerol and were frozen at -70 ℃. All strains were maintained on MH agar plates or nutrient agar, and antibacterial assays were conducted using MH broth. Bacterial growth was monitored as a function of turbidity by measuring optical density (OD) at 600 nm[14,15].

2.3. Determination of minimum inhibitory concentration(MIC)

MICs were determined using the method described in the Clinical and Laboratory Standards Institute (CLSI)[16]. The RHE MIC determinations with MRSA were performed using 96-well microtiter plates. Briefly, RHE was dissolved in DMSO followed by MH broth (final DMSO concentration=15% v/v). The suspension was serially diluted using 15% DMSO in MH broth after which 108CFU/mL of an overnight bacterial culture was inoculated into each well. The final volume in each well was 150 μL, and the DMSO concentration was 5% (which has no in fluence on bacterial growth).The DMSO concentration used is extremely important, because high concentrations can in fluence the activity of RHE. The samples were then incubated at 37 ℃ for 24 h. Twenty microliters of 1 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (1 mg/mL) was added to the suspension and incubated at 37 ℃ for 30 min. Clear yellowish-colored wells indicated inhibition of microbial growth, whereas dark blue-colored wells indicated the absence of inhibition. The MIC was defined as the lowest concentration producing no visible growth, as observed through the naked eye[17].

2.4. Checkerboard dilution test

The checkerboard dilution test was used to measure the synergistic effects of RHE and OX. OX was mixed in MH broth and serially diluted with RHE. The bacterial concentration was adjusted to 1.5×107CFU/mL. Suppression of microbial growth was checked for after incubation for 24 h at 37 ℃. Each experiment was conducted in triplicates. The interaction between RHE and OX was determined using the fractional inhibitory concentration index.

2.5. Reverse transcriptase polymerase chain reaction (PCR)

MRSA (ATCC 33591) was cultured in MH broth until an OD600 of 0.35-0.45 was reached; the culture was then treated with various concentrations of OX and RHE and incubated for 20 min at 37 ℃.RNA was extracted using the E.Z.N.A.? bacterial RNA kit (Omega Bio-tek, Norcross, GA, USA), in accordance with the manufacturer′s instructions. Single-strand cDNA synthesis was performed using the Quantitact Reverse Transcription Kit, in accordance with the manufacturer′s instructions. PCR was performed using the Maxime PCR PreMix (20 μL; iNtRon Biotechnology, Inc., Seongnam,Korea), primers, cDNA sample, and sterile distilled water. Primer sequences ofmecA,mecI,mecR1,blaZ,blaI,blaR1, and16s rRNAare presented in Table 1. After PCR, equal amounts of the PCR products(12 μL) were subjected to 1.5% agarose gel electrophoresis; bands were visualized under ultraviolet light.

Table 1 RT-PCR primer sequences.

2.6. Statistical analyses

The statistical analysis was assessed using one-way analysis of variance, followed by Scheffe′s test for multiple comparisons. Data were presented as means±standard deviations. All calculations were assessed using SPSS statistics 23 software (SPSS Inc.Chicago, IL,USA).P＜0.05 was considered statistically signi ficant.

3. Results

3.1. Determination of antibacterial activity

To examine the antibacterial activity of RHE, AM, and OX,antibacterial susceptibility testing was conducted on MRSA using the CLSI method. AM and OX, which are β-lactam antibiotics capable of inhibiting bacterial cell wall synthesis, were used as positive controls. RHE exhibited similar MICs in all tested strains.The RHE MIC values of all tested strains (ATTC and clinical isolates) ranged from 62.5 to 250.0 μg/mL (62.5 μg/mL for ATCC 33591), whereas AM MICs ranged from 0.97 to 125.00 μg/mL. The MIC for OX against ATCC 33591 was 1 000 μg/mL; MIC values ranged from 250 to 1 000 μg/mL for other strains. The results of the MIC assay are presented in Table 2.

Table 2 MICs of ampicillin, oxacillin, and rhein against various S. aureus strains (μg/mL).

3.2. Synergistic effects of RHE/antibiotic combinations against MRSA

A checkerboard assay was conducted to investigate the antibacterial effect of RHE combined with OX against MRSA. The combination of RHE and OX indeed exhibited synergistic effects against ATCC33591 and DHCR-1 and partial synergistic effects against DHCR-2 (Table 3).

Table 3 Synergy effects of rhein combined the β-lactam antibiotic oxacillin against S.aureus strains.

3.3. Polymerase chain reaction (PCR) analysis

RT-PCR was performed to evaluate the inhibitory effects of RHE against the MRSA resistance genes. RHE decreased the expression ofmecA/blaZin a dose-dependent manner. In contrast,their expression was increased by OX and again decreased with a combination of RHE and OX. In addition, the expression of the regulator genesmecI/blaIandmecR1/blaR1also decreased in the presence of RHE or RHE combined with OX (Figure 2 and 3).

Figure 2. Expression of PBP2a-related genes.

4. Discussion

Figure 3. Expression of β-lactamase-related genes.

Drug resistant bacteria are a menace to the healthcare system,because they limit remedies to infections worldwide. Resistance can occur through the acquisition of the resistance gene by horizontal gene transfer or through spontaneous mutation. BothmecAandblaZplay an important role in the β-lactam resistance of MRSA. The chromosomal genemecAencodes PBP2a, an alternative penicillinbinding protein having low affinity for β-lactams; PBP2a takes over the function of other PBPs that have been inactivated by β-lactam antibiotics[18]. TheblaZgene encodes β-lactamase, which inactivates penicillin by hydrolysis of the β-lactam ring[19]. Given restricted remedy in the research of novel antibacterial agent, there is notable study in targeting resistance genes to help re-establish susceptibility to commercial antibiotics[20]. Several researchers have previously reported that some antibacterial substances target these two β-lactam resistance genes,mecAandblaZgenes[21,22]. Therefore, we performed the study on antibacterial compound targeting β-lactam resistance genes. A previous study described the synergistic effect of RHE combined with AM or OX against MRSA[13]. We hypothesized that RHE produces a synergistic effect with OX in MRSA, because RHE suppresses β-lactam resistance genes. The role of PBP2a and β-lactamase are important in β-lactam resistance, and these proteins are expressed by the transcription ofmecAandblaZ, respectively.In the presence of OX, acylation ofBlaR1/MecR1, a sensor protein located in the cell membrane, is followed by the autoproteolytic cleavage of its cytoplasmic domain. The intracellular fragment ofBlaR1/MecR1travels to the SCCmec and proteolytically cleaves the BlaI/MecI repressor bound to the conserved TACA/TGTA DNA motif, thereby leading to the transcription of the mec/bla system[5-7].When RHE was added to the media containing MRSA, the gene expressions ofmecA/mecI/mecR1andblaZ/blaI/blaR1reduced. This suggests that RHE decrease β-lactam resistance of MRSA, because it inhibitsmecandblagenes located in SCCmec. When MRSA senses the presence of β-lactam antibiotics by MecR1/BlaR1, it produces resistance factors. The mec/bla genes expressions were increased by OX, again decreased with a combination of RHE and OX. It showed that RHE in fluences the MecR1/blaR1 located in the cell membrane of MRSA, thereby ultimately inhibiting the mec/bla system by interrupting signal transduction for transcription. Similar findings have been reported in a previous study wherein MRSA strains were treated with thioridazine, which affects membrane stability and induces conformational changes in theMecR1andBlaR1protein,thereby resulting in a subsequent decrease in the level of PBP2a activityviathe inhibition ofmecA/mecI/mecR1andblaZ/blaI/blaR1gene expression[21]. Chlorpromazine also was reported to have a similar effect. Chlorpromazine inhibits the expression ofmecA/mecI/mecR1andblaZ/blaI/blaR1by affecting the cell membrane of MRSA[23]. In conclusion, RHE inhibited the gene expressions of mec/bla system because of affecting theMecR1/BlaR1located in cell membrane of MRSA, which manifested itself as a synergistic effect between RHE and OX against MRSA. These results indicate that RHE was targeted to the expression of the resistance gene of MRSA.This study therefore demonstrates a new pharmacological activity for RHE, which could be a good candidate for clinical development as an antibacterial drug. The promising results of this study are expected to enhance the use of natural products as drugs in the future.

Conflicts of interest statement

The authors declare that there is no con flict of interest.