Transcriptome analysis of methicillin-resistant Staphylococcus aureus in response to stigmasterol and lupeol
Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) is a substantial global public health treat that causes morbidity and mortality worldwide [1] as well as increased healthcare costs [2]. It causes a diverse range of clinical diseases, ranging from benign superficial skin infections to severe life-threatening conditions such as bacteraemia, endocarditis, pneumonia, abscesses, and soft or bone-tissue infections [3]. Infections caused by MRSA are particularly difficult to treat because of the emergence of resistance to commonly used antibiotic classes and the limited therapeutic options [4]. Glycopeptide antibiotics such as vancomycin and teicoplanin are still the drugs of choice for serious MRSA infections, despite reports of glycopeptide-resistant MRSA isolates [5]. Linezolid, quinupristin/dalfopristin, daptomycin and telavancin are new antibiotics for drug-resistant bacteria [6] but the emergence of resistance to these agents [7], [8], [9] has limited their effectiveness. Thus, there is an urgent need for new antibiotics to combat the emergence of drug-resistant MRSA.
The emergence of micro-organisms resistant to synthetic antibiotics has focused the search on antimicrobial agents from plants. Currently, at least 119 chemicals have been derived from 90 plant species, which can be considered as important antibiotics in most countries [10].
Stigmasterol and lupeol from Phyllanthus columnaris stem bark were revealed to possess antibacterial activity against 16 MRSA strains and were also proven to be non-toxic to Vero cells [11]. Morphological and ultrastructural alterations of MRSA treated with both compounds after 24 h were revealed by scanning and transmission electron microscopy. Both compounds affected the cell wall and led to cell wall disruption, the release of cytoplasmic contents and decreased cellular volume [11]. Although both compounds have potential as new sources for the treatment of MRSA infections, the response mechanisms of MRSA to these compounds are still poorly understood.
This study implemented a next-generation sequencing (NGS) approach to determine the expression of MRSA essential genes under treatment with both compounds. To the best of our knowledge, this study represents the most comprehensive NGS study to date profiling the novel MRSA response to both compounds derived from P. columnaris. In comparison with previous studies, we have revealed morphological and ultrastructural alterations of the treated MRSA by electron microscopy, allowing us to further explore the statistically significant changes in gene expression in response to both bioactive compounds.
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Bacterial strains and materials
The MRSA strains used in this study were the reference strain ATCC 43300 and three clinical MRSA isolates (WM3, BM1 and KJ7). S. aureus ATCC 25923 was also included as control strain in the antibacterial testing. All strains were maintained on brain–heart infusion agar slopes (Oxoid Ltd., Basingstoke, UK) and were enriched in brain–heart infusion broth (Oxoid Ltd.) prior to use [12]. Mueller–Hinton agar (Oxoid Ltd.) and Mueller–Hinton broth (Oxoid Ltd.) were used as growth media for
Growth profile of MRSA exposed to stigmasterol and lupeol
According to the CLSI [13], bactericidal activity is defined as a ≥3 log10 CFU/mL reduction in the overall colony count from the initial inoculum. Bacteriostatic activity is defined as a <3 log10 CFU/mL reduction in the overall colony count from the initial inoculum.
The effect of stigmasterol exposure for 24 h on the growth of MRSA strains ATCC 43300, WM3, BM1 and KJ7 is as shown in Fig. 1. Stigmasterol exhibited an inhibitory effect only at 1× MIC against all tested MRSA strains and failed to
Antibacterial activity of lupeol and stigmasterol on MRSA
Both compounds exhibited an MIC of 12.5 mg/mL and an MBC >12.5 mg/mL against all MRSA tested, except for lupeol that showed MICs of 6.25 mg/mL on MRSA ATCC 43300 and KJ7 and an MBC of 12.5 mg/mL against BM1. Time–kill analysis showed that stigmasterol at 1× MIC inhibited all clinical strains from the beginning of exposure (0 h), followed by ATCC 43300 after 12 h. Lupeol inhibited ATCC 43300 and KJ7 at 6.25 mg/mL and 12.5 mg/mL after 12 h, and inhibition on WM3 with 12.5 mg/mL was shown after 4 h of
Conclusion
Both stigmasterol and lupeol exhibited a bacteriostatic effect against MRSA, except for lupeol that possessed both a bacteriostatic and bactericidal effect within the same MIC and MBC values on BM1 (12.5 mg/mL). NGS has allowed the detection of numerous essential antibacterial targets for further analysis and evaluation. To the best of our knowledge, no published data are available regarding the anti-MRSA effects of stigmasterol and lupeol compounds at the molecular level. Generally, both
Funding
This work was supported by the Ministry of Health Malaysia [grant no. NMMR-11-59-8309].
Ethical approval
Not required.
References (38)
- et al.
Serious infections due to methicillin-resistant Staphylococcus aureus: an evolving challenge for physicians
Eur J Intern Med
(2009) Microbiology of drugs for treating multiply drug-resistant Gram-positive bacteria
J Infect
(2009)- et al.
Staphylococcus aureus infections: new challenges from an old pathogen
Enferm Infecc Microbiol Clin
(2006) - et al.
Kinetics of antimicrobial activity
J Pediatr
(1986) - et al.
Effect of schedule of administration on the therapeutic efficacy of penicillin: importance of the aggregate time penicillin remains at effectively bactericidal levels
Am J Med
(1950) - et al.
RNA integrity and the effect on the real-time qRT-PCR performance
Mol Aspects Med
(2006) - et al.
Selection and validation of candidate housekeeping genes for studies of human keratinocytes—review and recommendations
J Invest Dermatol
(2009) - et al.
Control of bacterial transcription, translation and replication by (p)ppGpp
Curr Opin Microbiol
(2008) - et al.
Crystal structure of the bovine mitochondrial elongation factor Tu·Ts complex
J Biol Chem
(2005) - et al.
Invasive methicillin-resistant Staphylococcus aureus infections in the United States
JAMA
(2007)