Antimicrobial effect and mode of action of terpeneless cold-pressed Valencia orange essential oil on methicillin-resistant Staphylococcus aureus


Steven C. Ricke and Arunachalam Muthaiyan, Center for Food Safety-Department of Food Science, University of Arkansas, Fayetteville, AR 72704, USA. E-mails: or


Aims:  The objectives of this study were to evaluate the antistaphylococcal effect and elucidate the mechanism of action of orange essential oil against antibiotic-resistant Staphylococcus aureus strains.

Methods and Results:  The inhibitory effect of commercial orange essential oil (EO) against six Staph. aureus strains was tested using disc diffusion and agar dilution methods. The mechanism of EO action on MRSA was analysed by transcriptional profiling. Morphological changes of EO-treated Staph. aureus were examined using transmission electron microscopy. Results showed that 0·1% of terpeneless cold-pressed Valencia orange oil (CPV) induced the cell wall stress stimulon consistent with the inhibition of cell wall synthesis. Transmission electron microscopic observation revealed cell lysis and suggested a cell wall lysis–related mechanism of CPV.

Conclusions:  CPV inhibits the growth of Staph. aureus, causes gene expression changes consistent with the inhibition of cell wall synthesis, and triggers cell lysis.

Significance and Impact of the Study:  Multiple antibiotics resistance is becoming a serious problem in the management of Staph. aureus infections. In this study, the altered expression of cell wall-associated genes and subsequent cell lysis in MRSA caused by CPV suggest that it may be a potential antimicrobial agent to control antibiotic–resistant Staph. aureus.


In recent years, methicillin-resistant Staphylococcus aureus (MRSA) has appeared more in communities outside hospital settings and has emerged as a major public health concern worldwide (Kennedy et al. 2008; DeLeo et al. 2010). As infections caused by MRSA are increasing, as are rates of antibiotic therapy failures, new measures to treat and prevent this infectious pathogen are becoming inevitable (Pirri et al. 2009). One such approach to counter the antibiotic resistance emphasizes the search of biologically active pharmacophores possessing novel modes of action from natural resources (Saxena and Kumar 2002; Saleem et al. 2010). Natural products have been investigated and utilized to alleviate disease since early human history. Before the ‘synthetic era’, 80% of all medicines were obtained from roots, barks, leaves, flowers, seeds and fruits (McChesney et al. 2007).

Numerous studies have discovered promising novel antimicrobial candidates from plant-derived essential oils (EOs). EOs are particularly interesting as some oils have been used by native groups for curative purposes in the past (Saravolatz et al. 1982; Burt 2004). Also, research data indicate that many EOs have antimicrobial activity. For instance, tea tree oil obtained from Melaleuca alternifolia has been shown to be active against a wide range of micro-organisms (Gustafson et al. 1998; Hammer et al. 2006). In previous studies, the antimicrobial activities of other EOs have also been investigated, and their actions against various pathogens, including clinical MRSA isolates, have been demonstrated (Cox et al. 1998; Elsom and Hide 1999; Hammer et al. 1999; May et al. 2000; Takarada et al. 2002; Edwards-Jones et al. 2004; Brady et al. 2006; Prabuseenivasan et al. 2006; Chao et al. 2008; Doran et al. 2009; Tohidpour et al. 2010). There are also several clinical studies and case reports noting the successful use of EOs in treating MRSA nasal carriage and wound infections (Caelli et al. 2000; Sherry et al. 2001; Dryden et al. 2004; Sherry and Warnke 2004).

Fisher and Phillips (2006) studied the effectiveness of citrus EOs and their components citral, limonene and linalool against a number of common foodborne pathogens Listeria monocytenes, Staphylococcus aureus, Bacillus cereus, Escherichia coli O157 and Campylobacter jejuni both in vitro and on food models. Previous studies in our laboratory have demonstrated the inhibition of Salmonella (O’Bryan et al. 2008), E. coli O157: H7 (Nannapaneni et al. 2008), Listeria (Shannon et al. 2011) and Campylobacter (Nannapaneni et al. 2009) by citrus-derived cold-pressed Valencia orange oil, terpeneless Valencia orange oil, cold-pressed orange terpenes, high-purity orange terpenes, d-limonene, and terpenes from orange essence. However, these oils were not tested specifically against antibiotic-resistant Staph. aureus. Therefore, the objective of this study was to evaluate the inhibitory activity and mechanism of action of orange essential oil on Staph. aureus to determine their potential for use as antistaphylococcal agents against MRSA.

Materials and methods

Bacterial strains and growth conditions

The following Staph. aureus strains were used in this study: methicillin-susceptible strain SH1000 (Horsburgh et al. 2002), methicillin-resistant strains COL (Sabath et al. 1974), 13136 pm+ (Brown and Reynolds 1980), and N315 (Kuroda et al. 2001), and methicillin- and vancomycin intermediate–resistant strains 13136 pm+ V20 (Pfeltz et al. 2000), and Mu50 (Kuroda et al. 2001). Cultures were propagated in tryptic soya broth (TSB) (Difco Laboratories, Inc., Detroit, MI, USA). A loop of bacteria from a tryptic soya agar (TSA) (Difco Laboratories, Inc.) was inoculated into a 10-ml tube of sterile TSB and subsequently incubated for 18 h at 37°C, after which a 100-μl aliquot was transferred into a fresh sterile 10 ml of TSB, which was incubated an additional 18 h before use.

Orange essential oils

All EOs were obtained as commercially available products of Citrus sinensis (L.) Osbeck from Firmenich Citrus Center, Safety Harbor, FL, USA and were stored as per the manufacturer’s recommendations at 4°C prior to use. Oils tested included terpeneless cold-pressed Valencia orange oil (CPV), Valencia orange oil, cold-pressed orange terpenes, high-purity orange terpenes, d-limonene, terpenes from orange essence, fivefold concentrated Valencia orange oil and cold-pressed citronellal.

Disc diffusion assay for screening the inhibitory effect of EOs

Disc diffusion assay was carried out by the method described by O’Bryan et al. (2008). Overnight cultures of the Staph. aureus were streaked on sterile TSA (Difco Laboratories, Inc.) by dipping a sterile cotton swab into the culture. The swab was used to streak the agar plate to produce a lawn of growth by streaking the plate in three different directions. The orange EOs (10 μl) were aseptically pipetted onto sterile 6-mm paper discs (Becton Dickson, Franklin Lakes, NJ, USA), and subsequently, the paper discs were aseptically placed on the agar. Diameters of the zones of inhibitions were measured in millimetres after 24 h of incubation at 37°C. The assays were carried out on three independent experiments conducted in duplicate.

Minimum inhibitory concentration assay (MIC)

The MIC of CPV for Staph. aureus strains was performed using a modified agar dilution method. A final concentration of 0·5% (v/v) Tween-80 (Sigma, St Louis, MO, USA) was incorporated into the agar medium to enhance the oil solubility. Different concentrations of oil were added to the sterile TSA at 48°C. Plates were dried at room temperature for 12 h prior to spot inoculation with 5-μl aliquots of culture containing c. 5 log CFU per spot of each organism in triplicate. Inoculated plates were incubated at 37°C for 24 h, and the MICs were determined as the lowest concentration of oil inhibiting visible growth of organisms on the agar plate. Experiments were carried out in three independent experiments in duplicate. Inhibition of bacterial growth in the plates containing test oil was judged by comparing with the visible growth in control plates.

Growth inhibitory concentration (GIC) studies

Overnight grown cultures were used to inoculate (1% v/v) 20 ml TSB in 50-ml Erlenmeyer flasks and were grown at 37°C with shaking at 200 rev min−1. Two different concentrations (½ and 1× of MIC) of EO were used in this study. After adding the EO to the log-phase cultures (OD600, c. 0·4), growth was measured at 600 nm at regular intervals in a Beckman DU65 spectrophotometer. A final concentration of 0·5% (v/v) Tween-80 (Sigma) was used as a dispersing agent for EO.

RNA extraction and transcriptional profiling

On the basis of the GIC study, ½× MIC concentration of EO was added to the log-phase cells for 15 min of challenge. Control culture was not challenged with EO and was also incubated for 15 min. RNA extraction and microarray hybridization were carried out as described by Muthaiyan et al. (2008). Briefly, total bacterial RNA was extracted from 3 ml of culture which was mixed with 6 ml of bacterial RNA protect solution (Qiagen, Valencia, CA, USA) and subsequently centrifuged to collect the cells. To extract the RNA, bacterial pellets were resuspended in 1 ml of Trizol (Invitrogen, Grand Island, NY, USA), and the cells were broken using the FastPrep system (Qbiogene, Irvine, CA, USA) at a speed setting of 6·0 for 40 s. Extracted RNA was purified using the RNeasy mini kit (Qiagen). cDNA was generated from DNase-treated and purified RNA, using random hexamers (Invitrogen) as primers for reverse transcription. The primers were annealed (70°C for 10 min, followed by 1-min incubation in ice) to total RNA (2·5 μg) and were extended with SuperScript III reverse transcriptase (Invitrogen) with 0·1 mol l−1 dithiothreitol 12·5 mmol dNTP/aa-UTP (Ambion, Austin, TX, USA) mix at 42°C. Residual RNA was removed by alkaline treatment followed by neutralization, and cDNA was purified with a QIAquick PCR purification kit (Qiagen). Purified aminoallyl-modified cDNA was subsequently labelled with Cy3 or Cy5 monofunctional NHS ester cyanogen dyes (GE Healthcare, Piscataway, NJ, USA), according to the manufacturer’s instruction. Labelled cDNA was purified using a QIAquick PCR purification kit (Qiagen), and the purified labelled cDNA was hybridized with Staph. aureus genome microarrays version 6.0 provided by the Pathogen Functional Genomics Resource Center (PFGRC).

Microarray data analysis

Hybridization signals were scanned using an Axon4000B scanner with Acuity 6.0 software (Molecular Devices, Inc., Sunnyvale, CA, USA) and scans were saved as TIFF images. Data were analysed using tm4 microarray software suite (Saeed et al. 2003). Scans were analysed using tm4-Spotfinder software, and the local background was subsequently subtracted. The data set was normalized by applying the LOWESS algorithm using tm4-midas software. The normalized log2 ratio of test/control signal for each spot was recorded. Significant changes of gene expression were identified with significance analysis of microarrays (sam) software using one class mode (Tusher et al. 2001). The differentially regulated genes were further classified according to the functional categories described in the comprehensive microbial resource of TIGR ( As per our standard transcriptional profiling protocol, to minimize the technical and biological variations and to ensure that the data obtained were of good quality, three independent cultures were used to prepare RNA samples, and each RNA preparation was used to make probes for at least two separate arrays for which the incorporated dye was reversed (Muthaiyan et al. 2008).

Microarray data accession number

The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE33465 ( (Edgar et al. 2002).

Electron microscopy

TSB-grown exponential-phase Staph. aureus COL was treated with ½× MIC of the CPV for 30 min. Following the treatment, 2 ml of the culture was collected by centrifugation at 12 000 g for 10 min. The cell pellets were subsequently fixed in Karnovsky’s fixative for 2 h under a weak vacuum. Samples were rinsed three times in 0·05 mol cacodylate buffer, pH 7·2, postfixed in 1% osmium tetroxide (aqueous), rinsed with distilled water, and stained with 0·5% uranyl acetate overnight at 4°C. The sample was dehydrated in a graded ethanol series, followed by propylene oxide, and embedded in Spurr’s medium. Ultrathin sections were cut using a diamond knife on a MT2B Ultratome (Dupont Company, Newtown, CT, USA). The sections were placed on 300-mesh copper grid and stained with 2% aqueous uranyl acetate, followed by lead citrate. Grids were viewed at 80 kv using a JEM 100 CX transmission electron microscope (JEOL, Tokyo, Japan).


Inhibitory effect of citrus oils against Staphylococcus aureus

Of the eight EOs tested, terpeneless cold-pressed Valencia orange oil (CPV) and cold-pressed citronellal exhibited a high-level inhibition against all Staphaureus strains in disc diffusion assays (Table 1). The MICs of CPV for six Staph. aureus strains were determined using agar dilution method. CPV concentration at 0·18% caused complete inhibition for the strains 13136 pm+ and 13136 pm+V20. However, for strains COL, Mu50 and N315, 0·21% CPV was required to inhibit the growth. On the basis of the MICs, growth inhibitory concentration was determined for CPV to choose the concentration and duration of treatment for transcriptional profiling studies. Two different concentrations (½× and 1× of MIC) of CPV were used to determine the GIC. Both ½× and 1× of MIC concentrations caused significant growth inhibition for strains SH1000, COL, 13136 pm+, Mu50 and N315. However, the VISA strain 13136 pm+V20 exhibited reduced susceptibility to ½× MIC of CPV (Fig. 1).

Table 1.   Inhibitory effect of terpeneless cold-pressed Valencia orange oil (CPV) and citronellal against Staphylococcus aureus strains determined by a disc diffusion assay
Staph. aureus strainInhibition zone (mm)a
  1. *Inhibition zones are average values of three independent trials ± the standard deviation (SD, n = 6) of the mean.

SH100031·50 ± 3·029·20 ± 0·84
COL65·83 ± 3·7619·83 ± 1·33
13136 pm+65·67 ± 4·5918·83 ± 1·33
13136 pm+V2076·67 ± 4·0811·00 ± 1·26
N31565·83 ± 3·7611·17 ± 0·98
Mu5032·50 ± 2·748·33 ± 0·82
Figure 1.

 Growth inhibitory effect of CPV on Staphylococcus aureus strain SH1000 (a), COL (b), 13136 pm+ (c), 13136 pm+V20 (d), Mu50 (e), and N315 (f). ⋄, control; △, 1× MIC; □, ½× MIC.

Effect of CPV on the cell lysis–related gene expression

Staphylococcus aureus COL challenged with 0·1% CPV for 15 min showed upregulation of 431 genes and downregulation of 551 genes from a variety of functional categories (Tables S1 and S2). In the initial growth study, the CPV-treated COL cells showed rapid lysis within 60 min of the treatment; also, 24-fold induced expression of cwrA (SACOL2571) in the transcriptional profile supported the CPV-induced cell wall damage. Therefore, we particularly focused on the altered expression pattern of the cell wall-related genes to better understand the mechanism of the CPV action on Staph. aureus. In the transcriptional profiling analysis, about 62 and 36 cell envelope–related genes were under- and over-expressed, respectively. Some of the well-recognized cell wall stress stimulon member genes including penicillin-binding protein pbp1, pbp2(mecA), pbp3, and murein sacculus and peptidoglycan biosynthesis–related murB, murC, muD, murE, murG and autolysin-related atl, lytM were downregulated (−3- to −2-fold). pbp4 and capsular polysaccharide biosynthesis–related genes (cap) were upregulated in the cell envelope–related category (Table 2).

Table 2.   Altered expression of genes associated with cell lysis in CPV-treated Staphylococcus aureus COL cells
Locus IDGene*Gene/protein nameSubfunctional categoryFold change
 Cell envelope
Downregulated genes 
 SACOL0938dltDDltD proteinBiosynthesis and degradation of surface polysaccharides and lipopolysaccharides−10·41
 SACOL0054NAMur ligase family protein, authentic frameshiftBiosynthesis and degradation of murein sacculus and peptidoglycan−9·61
 SACOL0937dltCd-alanyl carrier proteinBiosynthesis and degradation of surface polysaccharides and lipopolysaccharides−5·99
 SACOL0936dltBDltB protein −5·41
 SACOL0247lrgAHolin-like protein LrgABiosynthesis and degradation of murein sacculus and peptidoglycan−4·64
 SACOL0052NAGlycosyl transferase, group 1 family proteinBiosynthesis and degradation of surface polysaccharides and lipopolysaccharides−4·19
 SACOL1195mraYPhospho-N-acetylmuramoyl-pentapeptide-transferaseBiosynthesis and degradation of murein sacculus and peptidoglycan−3·71
 SACOL0697tagXTeichoic acid biosynthesis protein X −3·51
 SACOL0801murBUDP-N-acetylenolpyruvoylglucosamine reductase −2·97
 SACOL1023murEUDP-N-acetylmuramoylalanyl-d-glutamate--2,6-diaminopimelate ligase −2·89
 SACOL1194pbp1Penicillin-binding protein 1 −2·5
 SACOL1196murDUDP-N-acetylmuramoylalanine--d-glutamate ligase −2·45
 SACOL2074NAd-alanine--d-alanine ligase −2·39
 SACOL1687NAN-acetylmuramoyl-l-alanine amidase, family 3Biosynthesis and degradation of surface polysaccharides and lipopolysaccharides−2·38
 SACOL0543glmUUDP-N-acetylglucosamine pyrophosphorylase −2·21
 SACOL2103NAUDP-N-acetylglucosamine 2-epimerase −2·21
 SACOL0033mecAPenicillin-binding protein 2Biosynthesis and degradation of murein sacculus and peptidoglycan−2·15
 SACOL1329femCGlutamine synthetase FemC −2·15
 SACOL0242NATeichoic acid biosynthesis protein, putative −2·12
 SACOL1134kdtBLipopolysaccharide core biosynthesis protein KdtBBiosynthesis and degradation of surface polysaccharides and lipopolysaccharides−2·09
 SACOL1609pbp3Penicillin-binding protein 3Biosynthesis and degradation of murein sacculus and peptidoglycan−2·01
 SACOL1453murGUDP-N-acetylglucosamine--N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase −1·98
 SACOL0263lytMPeptidoglycan hydrolase −1·96
 SACOL0695NAtagG protein, teichoic acid ABC transporter protein, putative −1·87
 SACOL1066NAfmt protein −1·83
 SACOL1062atlBifunctional autolysinBiosynthesis and degradation of surface polysaccharides and lipopolysaccharides−1·78
 SACOL1424NAPhosphate ABC transporter, phosphate-binding proteinOther−1·74
 SACOL1790murCUDP-N-acetylmuramate–alanine ligaseBiosynthesis and degradation of murein sacculus and peptidoglycan−1·58
 SACOL1951NAMur ligase family protein −1·57
Upregulated genes 
 SACOL2571cwrAConserved hypothetical proteinConserved24·05
 SACOL1434NAAlanine racemase family proteinBiosynthesis and degradation of murein sacculus and peptidoglycan21·26
 SACOL0147cap5LCapsular polysaccharide biosynthesis protein Cap5LBiosynthesis and degradation of surface polysaccharides and lipopolysaccharides4·22
 SACOL0150cap5OCapsular polysaccharide biosynthesis protein Cap5O 4·02
 SACOL0148cap5MCapsular polysaccharide biosynthesis galactosyltransferase Cap5M 3·98
 SACOL0151cap5PUDP-N-acetylglucosamine 2-epimerase Cap5P 3·61
 SACOL0149cap5NCapsular polysaccharide biosynthesis protein Cap5N 3·55
 SACOL1161murIGlutamate racemaseBiosynthesis and degradation of murein sacculus and peptidoglycan3·11
 SACOL0146cap5KCapsular polysaccharide biosynthesis protein Cap5KBiosynthesis and degradation of surface polysaccharides and lipopolysaccharides2·95
 SACOL0144cap5ICapsular polysaccharide biosynthesis protein Cap5I 2·83
 SACOL0145cap5JCapsular polysaccharide biosynthesis protein Cap5J 2·79
 SACOL0140cap5ECapsular polysaccharide biosynthesis protein Cap5E 2·67
 SACOL0141cap5FCapsular polysaccharide biosynthesis protein Cap5F 2·17
 SACOL2092murAAUDP-N-acetylglucosamine 1-carboxyvinyltransferase 1Biosynthesis and degradation of murein sacculus and peptidoglycan2·1
 SACOL0143cap5HCapsular polysaccharide biosynthesis protein Cap5HBiosynthesis and degradation of surface polysaccharides and lipopolysaccharides2·04
 SACOL0699pbp4Penicillin-binding protein 4Biosynthesis and degradation of murein sacculus and peptidoglycan2·04
 SACOL0142cap5GUDP-N-acetylglucosamine 2-epimerase Cap5GBiosynthesis and degradation of surface polysaccharides and lipopolysaccharides1·99
 SACOL0136cap5ACapsular polysaccharide biosynthesis protein Cap5A 1·68
 SACOL0137cap5BCapsular polysaccharide biosynthesis protein Cap5B 1·67
 SACOL2116murABUDP-N-acetylglucosamine 1-carboxyvinyltransferase 2Biosynthesis and degradation of murein sacculus and peptidoglycan1·67
 Cellular processes
Downregulated genes 
 SACOL0935dltAd-alanine-activating enzyme/d-alanine-d-alanyl carrier protein ligaseToxin production and resistance−11·93
 SACOL2295NAStaphyloxanthin biosynthesis protein, putativePathogenesis−8·55
 SACOL1535srrADNA-binding response regulator SrrA −8
 SACOL0244scdAScdA proteinCell division−6·24
 SACOL0270NAStaphyloxanthin biosynthesis protein, putativePathogenesis−5·55
 SACOL0095spaImmunoglobulin G–binding protein A precursor −5·54
 SACOL1437NAcold shock protein, CSD familyAdaptations to atypical conditions−4·87
 SACOL2126luxSAutoinducer-2 production protein LuxSOther−4·15
 SACOL0956kapBKinase-associated protein BAdaptations to atypical conditions−3·8
 SACOL1193NACell division protein FtsLCell division−3·6
 SACOL0608sdrCsdrC proteinCell adhesion−3·53
 SACOL1328glnRGlutamine synthetase repressorToxin production and resistance−3·35
 SACOL2291NAStaphyloxanthin biosynthesis proteinPathogenesis−3·33
 SACOL0552NAGeneral stress protein 13Adaptations to atypical conditions−3·32
 SACOL1534srrBSensor histidine kinase SrrBPathogenesis−3·27
 SACOL0743bacABacitracin resistance proteinToxin production and resistance−3·23
 SACOL1396fmtCfmtC protein −3·18
 SACOL2075ftsWCell division protein, FtsW/RodA/SpoVE familyCell division−3·12
 SACOL1197divIBCell division protein −2·91
 SACOL0766saeRDNA-binding response regulator SaeRPathogenesis−2·87
 SACOL1205NACell division initiation protein, putativeCell division−2·86
 SACOL0452ahpCAlkyl hydroperoxide reductase, C subunitDetoxification−2·8
 SACOL1537scpBSegregation and condensation protein BCell division−2·72
 SACOL1624eraGTP-binding protein Era −2·46
 SACOL1538scpASegregation and condensation protein A −2·31
 SACOL1184NAExfoliative toxin, putativeToxin production and resistance−2·14
 SACOL0746norRTranscriptional regulator, MarR family −2·06
 SACOL2731NACold shock protein, CSD familyAdaptations to atypical conditions−2·06
 SACOL1383mscLLarge conductance mechanosensitive channel protein −1·96
 SACOL2024agrDAccessory gene regulator protein DPathogenesis−1·91
 SACOL0610sdrEsdrE proteinCell adhesion−1·89
 SACOL1410femAfemA proteinToxin production and resistance−1·76
 SACOL0765saeSSensor histidine kinase SaeSPathogenesis−1·69
 SACOL0245lytSSensor histidine kinase LytS −1·66
 SACOL1198ftsACell division protein FtsACell division−1·53
Upregulated genes 
 SACOL1943vraSSensor histidine kinase VraSToxin production and resistance5·22
 SACOL1450arlSSensor histidine kinase ArlSPathogenesis4·74
 SACOL2353tcaRTranscriptional regulator TcaRToxin production and resistance4·18
 SACOL0672sarAStaphylococcal accessory regulator A 4·11
 SACOL2157NADrug resistance transporter, EmrB/QacA subfamily 3·75
 SACOL1451arlRDNA-binding response regulator ArlRPathogenesis3·61
 SACOL2289sarYStaphylococcal accessory regulator YToxin production and resistance3·21
 SACOL2258sarVStaphylococcal accessory regulator V 2·87
 SACOL1942vraRDNA-binding response regulator VraR 2·61
 SACOL1003NANegative regulator of competence MecA, putativeDNA transformation2·01
 SACOL0608sdrCsdrC proteinCell adhesion1·82
 Protein fate
Downregulated genes 
 SACOL1801NAPeptidase, M20/M25/M40 familyDegradation proteins, peptides and glycopeptides−3·45
 SACOL0806pepTPeptidase T −2·89
 SACOL2385NAHeat shock protein, Hsp20 familyProtein folding and stabilization−2·77
 SACOL1946NAMethionine aminopeptidase, type IProtein modification and repair−2·62
 SACOL1777NASerine protease HtrA, putativeDegradation proteins, peptides and glycopeptides−2·42
 SACOL0581secEPreprotein translocase, SecE subunitProtein and peptide secretion and trafficking−2·13
 SACOL1588NAProline dipeptidaseDegradation proteins, peptides and glycopeptides−2·12
 SACOL2038NAMetalloendopeptidase, putative, glycoprotease family −2·06
Upregulated genes 
 SACOL0979clpBATP-dependent Clp protease, ATP-binding subunit ClpBDegradation proteins, peptides and glycopeptides9·42
 SACOL1433NAPeptidase, M20/M25/M40 family 8·7
 SACOL2125NAPeptidase, M20/M25/M40 family 7·13
 SACOL0570clpCATP-dependent Clp protease, ATP-binding subunit ClpC, authentic frameshift 5·92
 SACOL1636dnaJdnaJ proteinProtein folding and stabilization4·94
 SACOL0833clpPATP-dependent Clp protease, proteolytic subunit ClpPDegradation proteins, peptides and glycopeptides3·72
 SACOL1638grpEHeat shock protein GrpEProtein folding and stabilization3·5
 SACOL0968spsASignal peptidase IA, inactiveProtein and peptide secretion and trafficking2·98
 SACOL1637dnaKdnaK proteinProtein folding and stabilization2·67
 SACOL2438NAEndopeptidase, putativeDegradation proteins, peptides and glycopeptides2·46
 SACOL1459NAPeptide methionine sulfoxide reductase, degenerateProtein modification and repair2·38
 SACOL2016groELchaperonin, 60 kDaProtein folding and stabilization2·36
 SACOL0556NAChaperonin, 33 kDa 2·17
 SACOL0844secGPreprotein translocase, SecG subunitProtein and peptide secretion and trafficking2·13
 SACOL0969spsBSignal peptidase IB 2·02
 SACOL2017groESChaperonin, 10 kDaProtein folding and stabilization1·81

Related to the cell envelope, classified under cellular processes, c. 54 and 49 genes were down- and upregulated, respectively. d-alanine-activating enzyme/d-alanine-d-alanyl carrier protein ligase encoding dltA, dltB, dltC, dltD; cell division-related divIB, ftsA, ftsL, ftsW; and universal stress resistance family protein encoding SACOL1753, SACOL1759, SACOL0552; and drug resistance transporter EmrB/QacA subfamily encoding SACOL2347 were downregulated (−11- to −2-fold). Two-component response regulators encoding vraS, vraR, arlS, arlR and transcriptional regulator tcaR and staphylococcal accessory regulator encoding sarA, sarV, sarY were upregulated between 2- and 5-fold (Table 2).

Another prominent category of genes altered by CPV treatment is the protein fate, a set of genes which include chaperones and proteases. Some of the genes encoding the chaperones and proteases are known as marker genes for cell wall stress condition. In this protein fate category 24, genes were downregulated and 26 genes were upregulated by CPV treatment. Most of the genes encoding for the degradation of proteins, peptides and glycopeptides were downregulated between −4- and −2-fold. However, CPV induced the expression of clpB and clpC (chaperone/protease), spsA and spsB (type 1 signal peptidases A and B), and SACOL2683 (putative methionine sulfoxide reductase). In addition, expression of genes encoding the major heat shock proteins GroEL, GroES, DnaK, DnaJ, GrpE had increased between 2- and 5-fold (Table 2).

In addition to the cell wall-related genes, a variety of genes involved in DNA metabolism that play a role in DNA replication, DNA recombination and DNA repair were down- and upregulated by CPV challenge (Table S1 and S2). Some of the known DNA metabolism–related genes affected by the CPV treatment encode DNA repair protein RecN, R subunit of type I restriction-modification enzyme, M subunit of type I restriction-modification enzyme, DNA gyrase, single-stranded DNA-specific exonuclease RecJ, ATP-dependent DNA helicase PcrA, DNA polymerase and DNA ligase.

Electron microscopic analysis of CPV-induced cell wall damage

Actively growing COL cells exhibited an ultrastructure typical of Staph. aureus, with septa that were normal in appearance and a trilaminar cell wall (Fig. 2a), whereas CPV (0·1%) treatment for 30 min led to extensive cell lysis in COL cells (Fig. 2b). In addition to the cell lysis, loss of cellular electron dense material, coagulation of cytoplasmic constituents, deformed septum and lack of a distinct midline were observed. As a consequence of profound structural alterations and breaks in the cell walls, several ghosts of lysed cells appeared after the CPV treatment (Fig. 2b). Electron microscopic observation of cell wall lysis in CPV-treated cells substantiates the results of CPV-induced altered expression of cell wall- and cell division-related genes in Staph. aureus.

Figure 2.

 Electron micrograph of ½× MIC of CPV-treated Staphylococcus aureus COL cells. a, control; b, cells after 30 min of CPV treatment. Magnification, ×30 000, 1 mm = 33·33 nm.


The infections caused by MRSA and VISA because of the acquisition of resistance towards current antimicrobials pose a serious challenge for therapy (Payne 2008). In an effort to explore the potential use of orange EO against antibiotic-resistant Staph. aureus, in this study inhibitory effects and mode of action of CPV against MRSA and VISA strains were studied. The inhibitory effect of CPV against each Staph. aureus strain varied in the disc diffusion screening assay. The VISA strain 13136 pm+ V20 exhibited greater inhibition (76·67 ± 4·08 mm) than other MRSA strains. Similar to our results, inhibitory effects of various EOs against MRSA and other bacteria have previously been confirmed using disc diffusion and agar dilution methods (Dorman and Deans 2000; Burt and Reinders 2003; Abulrob et al. 2004; Nostro et al. 2004; Fisher and Phillips 2006; Prabuseenivasan et al. 2006; Busatta et al. 2008; Chao et al. 2008; Viuda-Martos et al. 2008; Goñi et al. 2009; Patharakorn et al. 2010).

In previous reports, antimicrobial properties of EOs and their components have been reviewed extensively (Burt 2004). However, only a few studies have reported the mechanism of antibacterial action of EOs in great detail (Cox et al. 1998, 2000; Fisher and Phillips 2006). Therefore, based on the significant inhibitory effect we observed in our study, we selected CPV for the further experiments to study the mode of action on MRSA.

Numerous studies have investigated the changes in gene expression patterns in response to antibiotics at the subinhibitory concentration to obtain an in-depth understanding of the mode of action of antimicrobials (Wecke and Mascher 2011). Accordingly, in our study, Staph. aureus genome microarrays were used to capture the genomic response of CPV-treated Staph. aureus cells. Transcriptional profiling revealed alteration of gene expression in a variety of functional categories including amino acid biosynthesis, cell envelope, cellular processes, central intermediary metabolism, DNA metabolism, protein synthesis and signal transduction. Specifically, the observation of 24-fold induced expression of cwrA (SACOL2571) (equivalent locus SA2343 in strain N315) along with rapid lysis of Staph. aureus cells during the CPV treatment supported the CPV-induced cell wall damage in Staph. aureus. In previous research, high-level upregulation of cwrA has been reported in a variety of transcriptomic studies examining cell wall inhibition (McAleese et al. 2006; Sobral et al. 2007; Balibar et al. 2009), and recently, Balibar et al. (2010) showed that cwrA was robustly induced by cell wall-targeting antibiotics, vancomycin, oxacillin, penicillin G, phosphomycin, imipenem, hymeglusin and bacitracin, but not by antimicrobials with other mechanisms of action, including ciprofloxacin, erythromycin, chloramphenicol, triclosan, rifampicin, novobiocin and carbonyl cyanide 3-chlorophenylhydrazone. Therefore, we focused on the cell wall-related genes to elucidate the mechanisms of action of CPV. In support of our view in a similar study Cox et al. (2000) demonstrated that in E. coli and Staph. aureus, the antimicrobial activity of tea tree oil results from its ability to disrupt the permeability barrier of membrane structures. In a later study, Carson et al. (2002) reported that the mechanism of action of tea tree oil on Staph. aureus is not specific on cytoplasmic membrane but owing to induction of the release of membrane-bound cell wall autolytic enzymes and eventual cell lysis.

In earlier studies, based on the results of transcriptional profiling experiments after the exposure of Staph. aureus to cell wall-active agents, a set of cell wall-associated genes were identified and assigned as a ‘cell wall stimulon’. These genes have been used as marker genes for cell wall-related gene response (Kuroda et al. 2003; Utaida et al. 2003; Wilkinson et al. 2005; Gardete et al. 2006). In our study, we observed the altered expression of several cell wall stimulon member genes in CPV-treated Staph. aureus cells, and those genes are discussed in the following sections.

The PBPs are membrane-associated proteins that catalyse the final step of murein biosynthesis of cell wall peptidoglycan in Staph. aureus. PBP1 (pbp1) is essential and important for cell division (Pereira et al. 2007), and PBP2a, encoded by mecA, is responsible for methicillin resistance in Staph. aureus (Pinho and Errington 2005). It has been reported that inactivation of pbp3 caused a small but significant decrease in autolysis rates. Cells of abnormal size and shape and disoriented septa were reported when bacteria with inactivated pbp3 were grown in the presence of cell wall-active antibiotic methicillin (Pinho et al. 2000). In our study, downregulation of these PBPs encoding genes could very well be involved in the observed lysis of CPV-treated Staph. aureus cells. Additionally, we believe that the observed upregulation of PBP4 encoding pbp4 and capsular polysaccharide–related cap genes could be a protective response of Staph. aureus during the CPV-induced cell wall damage. In support of our view, it was recently demonstrated that PBP4 may play an important role in cell wall antibiotic resistance (Memmi et al. 2008; Navratna et al. 2010).

The Staph. aureus cell wall has been reported as a structure composed of highly cross-linked peptidoglycan, a complex structure composed of sugars and amino acids (murein), teichoic acids and cell wall-associated proteins (Dmitriev et al. 2004). We observed the repression of these murein sacculus and peptidoglycan biosynthesis–related murB, murC, muD, murE, murG, murAB genes upon the CPV treatment. Cell wall autolysis–related atl and lytM genes were also downregulated by CPV treatment. Downregulation of atl and lytM has previously been reported in Staph. aureus exposed to cell wall-active agents and viewed as a response of the cell to preserve peptidoglycan when challenged with cell wall-active agents (Kuroda et al. 2003; Utaida et al. 2003; Wilkinson et al. 2005; Muthaiyan et al. 2008).

Along with cell envelope–related group, genes belonging to Staph. aureus cellular processes were affected by CPV treatment. d-alanine-activating enzyme/d-alanine-d-alanyl carrier protein ligase encoding dltA, dltB, dltC, dltD was reportedly downregulated in CPV-treated cells. dltABCD operon controls the alanylation of wall teichoic acids which are involved in the control of autolysin activity in Staph. aureus. Previous research by Peschel et al. (1999, 2000) demonstrated that mutation in dlt genes leads to the failure of alanylation in the teichoic acids, and consequently, Staph. aureus cells become sensitive to human defensin HNP1–3, animal-derived protegrins, tachyplesins and magainin II; the bacteria-derived peptides gallidermin and nisin; and cell wall antibiotics. Thus, the repression of dlt genes by the CPV could be viewed as one of the reasons for the rapid lysis of CPV-treated cells.

Genes involved in the cell division and stress resistance were also downregulated in the CPV-treated cells. In previous transcriptional profiling studies, cell division proteins encoding genes were shown to be induced by cell wall antibiotics but downregulated by membrane-active compounds (Muthaiyan et al. 2008). Downregulation of these genes in response to CPV treatment indicates that CPV potentially acts on both cell walls and membranes of the Staph. aureus cells.

In Staph. aureus, vraSR is a two-component system that positively regulates a number of genes involved in cell wall synthesis, and arlSR is an another two-component system involved in several cell wall activities including rate of autolysis as well as the attachment to a polymer (Fournier and Hooper 2000; Kuroda et al. 2003). Induction of these two component response regulators along with arlSR-associated accessory regulators sarA, sarV, sarY in response to CPV treatment could be contemplated as a Staph. aureus protective response to the cell wall damage caused by the downregulation of cell wall synthesis–associated genes.

Some of the known cell wall stress stimulon genes encoding the chaperones and proteases were affected during exposure to CPV. Electron microscopic analysis of CPV-treated cells revealed that CPV acted on Staph. aureus cell walls and caused profound cell wall damage and cell lysis. Treatment of Staph. aureus cells with cell wall-active agents is considered to result in the accumulation of misfolded and damaged proteins (Utaida et al. 2003; Wilkinson et al. 2005; Muthaiyan et al. 2008). Presumably in an attempt to counter the CPV-mediated peptidoglycan biosynthesis inhibition and subsequent cell lysis, Staph. aureus increased the chaperones to restore the lysed proteins. Therefore, we speculate that the genes associated with the degradation of proteins and peptides clpB and clpC (chaperone/protease), spsA and spsB (type 1 signal peptidases A and B), and SACOL2683 (putative methionine sulfoxide reductase), and genes encoding the major heat shock proteins GroEL, GroES, DnaK, DnaJ, GrpE were induced in CPV-treated cells.

In addition to the cell wall-related genes, DNA replication-, DNA recombination- and DNA repair-related genes were also apparently down- and upregulated in CPV-treated cells. In previous studies, some of these genes have been identified as members of the cell wall stress stimulon. In Staph. aureus, altered expression of genes involved in DNA metabolism has been recognized as a characteristic mode of cell wall-active agent’s action and reported as a part of SOS response (Maiques et al. 2006). Also, in E. coli, it has been reported that transcription of genes encoding DNA polymerase and DNA repair is induced during the inhibition of cell wall synthesis caused by ß-lactam antibiotics (Perez-Capilla et al. 2005). Interestingly, in our study, c. 31 genes related to DNA replication and repair were repressed and 20 genes were upregulated upon CPV treatment. Therefore, we speculate that along with cell wall damage, CPV may possibly repress the Staph. aureus SOS system that normally used by the bacterial cells to survive during the adverse condition.

The electron microscopic observation of cell morphology of CPV-treated cells confirmed the transcriptional response of CPV-treated Staph. aureus cells exhibiting downregulation of cell envelope–related genes and corroborated the cell wall-active and lytic effect of CPV on Staph. aureus. Electron micrographs illustrated the cell wall and membrane damage and loss of cytoplasmic materials and several cell wall ghosts that accompanied in CPV treatment of Staph. aureus cells. Similar cytoplasmic losses have also been reported in tea tree oil–treated Staph. aureus cells (Carson et al. 2002). Apparently the downregulation of the autolysis-related gene observed in the transcriptional profiling may serve as a protective response of cells from the CPV-mediated cell wall lysis observed in the electron microscope.

Results of this in vitro study indicate that CPV effectively inhibits the Staph. aureus by affecting the cell wall. While the MRSA is becoming a significant public health problem, the findings of the present study are promising and reveal the potential of CPV as an alternative natural therapeutic antimicrobial agent against MRSA. However, prior to the use of CPV for MRSA decolonization, issues of safety and toxicity will need to be addressed.


The Staphylococcus aureus microarrays were obtained through NIAID’s Pathogen Functional Genomics Resource Center; managed and funded by Division of Microbiology and Infectious Diseases, NIAID, NIH, DHHS; and operated by The Institute for Genomic Research (TIGR). B.J.W. was supported by National Institute of Health (BJW NIH 1R15AI084006).