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. Author manuscript; available in PMC: 2013 May 1.
Published in final edited form as: J Appl Microbiol. 2012 Mar 20;112(5):1020–1033. doi: 10.1111/j.1365-2672.2012.05270.x

Antimicrobial Effect and Mode of Action of Terpeneless Cold Pressed Valencia Orange Essential Oil on Methicillin-Resistant Staphylococcus aureus

Arunachalam Muthaiyan 1,*, Elizabeth M Martin 1, Senthil Natesan 2, Philip G Crandall 1, Brian J Wilkinson 3, Steven C Ricke 1,*
PMCID: PMC3324624  NIHMSID: NIHMS359571  PMID: 22372962

Abstract

Aims

The objective of this study was to evaluate the antistaphylococcal effect and elucidate the mechanism of action of orange essential oil against antibiotic resistant Staphylococcus aureus strains.

Methods and Results

Inhibitory effect of commercial orange essential oil (EO) against six S. aureus strains was tested by disc diffusion and agar dilution methods. The mechanism of EO action on MRSA was analyzed by transcriptional profiling. Morphological changes of EO treated S. aureus were examined by transmission electron microscopy. Results showed that 0.1% of cold pressed terpeneless Valencia orange oil (CPV) induced the cell wall stress stimulon consistent with 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 S. aureus, causes gene expression changes consistent with 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 S. aureus infections. In this study the altered expression of cell wall associated genes and subsequent cell lysis in MRSA caused by CPV suggests that it may be a potential antimicrobial agent to control antibiotic resistant S. aureus.

Keywords: Staphylococcus aureus, MRSA, Natural antimicrobial, Mechanism of Essential Oil

Introduction

In recent years methicillin-resistant S. aureus (MRSA) has appeared more in communities outside the hospital settings and has emerged as a major public health concern worldwide (Kennedy et al. 2008, DeLeo et al. 2010). Since 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 since 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 microorganisms (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 (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, S. 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), Escherichia 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 S. aureus. Therefore, the objective of this study was to evaluate the inhibitory activity and mechanism of action of orange essential oil on S. aureus to determine their potential for use as antistaphylococcal agents against MRSA.

MATERIALS AND METHODS

Bacterial strains and growth conditions

The following S. aureus strains were used in this study: methicillin-susceptible strain SH1000 (Horsburgh et al. 2002), methicillin-resistant strains COL (Sabath et al. 1974), 13136 p−m+ (Brown and Reynolds 1980), and N315 (Kuroda et al. 2001), and methicillin- and vancomycin intermediate-resistant strains 13136 p−m+ V20 (Pfeltz et al. 2000), and Mu50 (Kuroda et al. 2001). Cultures were propagated in tryptic soya broth (TSB) (Difco Laboratories, Inc. Detroit, MI). 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 essential oils were obtained as commercially available products of Citrus sinensis (L.) Osbeck from Firmenich Citrus Center, Safety Harbor, FL, USA and were stored per 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, 5-fold 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. (O’Bryan et al. 2008). Overnight cultures of the S. 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 3 different directions. The orange EOs (10 μL) were aseptically pipetted onto sterile 6-mm paper discs (Becton Dickson, Franklin Lakes, NJ) and subsequently the paper discs were aseptically placed on the agar. Diameters of zones of inhibitions were measured in mm 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 S. aureus strains was performed by modified agar dilution method. A final concentration of 0.5% (v/v) Tween-80 (Sigma, St. Louis, MO) was incorporated into the agar medium to enhance 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 approximately 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 comparison with 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 rpm. Two different concentrations (½, and 1x of MIC) of EO were used in this study. After adding the EO to the log phase cultures (OD600 approximately 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

Based on the GIC study ½ x 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 was 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) and subsequently centrifuged to collect the cells. To extract the RNA, bacterial pellets were resuspended in 1 ml of Trizol (Invitrogen, Grand Island, NY) and the cells were broken using the FastPrep system (Qbiogene, Irvine, CA) at a speed setting of 6.0 for 40 seconds. Extracted RNA was purified using the RNeasy mini kit (Qiagen). cDNA was generated from DNase treated and purified RNA by 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 M dithiothreitol 12.5 m mol dNTP/aa-UTP (Ambion, Austin, TX) 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 labeled with Cy3 or Cy5 mono-functional NHS ester cyanogen dyes (GE Healthcare, Piscataway, NJ) according to the manufacturer’s instruction. Labeled cDNA was purified using a QIAquick PCR purification kit (Qiagen) and the purified labeled cDNA was hybridized with S. 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. U.S.) and scans were saved as TIFF images. Data analysis was performed by TM4 microarray software suite (Saeed et al. 2003). Scans were analyzed 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 (http://cmr.tigr.org/tigr-scripts/CMR/shared/Genomes.cgi). 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 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE33465) (Edgar et al. 2002).

Electron microscopy

TSB grown exponential phase S. aureus COL was treated with ½ x MIC of the CPV for 30 min. Following the treatment, 2 mL of the culture was collected by centrifugation at 10,000 RPM for 10 min. The cell pellets were subsequently fixed in Karnovsky’s fixative for 2 h under a weak vacuum. Samples were rinsed 3 times in 0.05 mol cacodylate buffer, pH 7.2, post fixed 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. Ultra-thin sections were cut with a diamond knife on a MT2B Ultratome (Dupont Company, Newtown, CT). 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 with a JEM 100 CX transmission electron microscope (JEOL, Tokyo, Japan).

RESULTS

Inhibitory effect of citrus oils against S. aureus

Of the eight EOs tested terpeneless cold pressed Valencia orange oil (CPV) and cold pressed citronellal exhibited a high level inhibition against all S. aureus strains in disc diffusion assays (Table 1). The MICs of CPV for six S. aureus strains were determined by agar dilution method. CPV concentration at 0.18% caused complete inhibition for the strains 13136 p−m+ and 13136 p−m+V20. However, for strains COL, Mu50, and N315 0.21% CPV was required to inhibit the growth. Based on 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 x and 1 x of MIC) of CPV were used to determine the GIC. Both ½ x and 1 x of MIC concentrations caused significant growth inhibition for strains SH1000, COL, 13136 p−m+, Mu50, and N315. However, the VISA strain 13136 p−m+V20 exhibited reduced susceptibility to ½ x MIC of CPV (Fig 1).

Table 1.

Inhibitory effect of terpeneless cold pressed Valencia orange oil (CPV) and citronellal against S. aureus strains determined by a disk-diffusion assay

S. aureus strain Inhibition Zone (mm)a
CPV Citronellal
SH1000 31.50 ± 3.02 9.20 ± 0.84
COL 65.83 ± 3.76 19.83 ± 1.33
13136 p−m+ 65.67 ± 4.59 18.83 ± 1.33
13136 p−m+V20 76.67 ± 4.08 11.00 ± 1.26
N315 65.83 ± 3.76 11.17 ± 0.98
Mu50 32.50 ± 2.74 8.33 ± 0.82
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a

Inhibition zones are average values of three independent trials ± the standard deviation (SD, n=6) of the mean.

Figure 1.

Figure 1

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Growth inhibitory effect of CPV on S. aureus strain SH1000 (a), COL (b), 13136 p−m+ (c), 13136 p−m+V20 (d), Mu50 (e), and N315 (f). ◇, control; △, 1x MIC; □, ½ x MIC.

Effect of CPV on the cell lysis related gene expression

S. aureus COL challenged with 0.1% CPV for 15 min showed upregulation of 431 genes and down regulation of 551 genes from a variety of functional categories (Supplementary Table 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 S. aureus. In the transcriptional profiling analysis about 62 and 36cell envelope-related genes were under- and over-expressed, respectively. Some of the well recognized cell wall stress stimulon member genesinclude 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 S. aureus COL cells

Locus ID Genea Gene/Protein Name Sub-functional Category Fold Change
Cell envelope
Downregulated Genes
SACOL0938 dltD DltD protein Biosynthesis and degradation of surface polysaccharides and lipopolysaccharides −10.41
SACOL0054 NA Mur ligase family protein, authentic frameshift Biosynthesis and degradation of murein sacculus and peptidoglycan −9.61
SACOL0937 dltC D-alanyl carrier protein Biosynthesis and degradation of surface polysaccharides and lipopolysaccharides −5.99
SACOL0936 dltB DltB protein −5.41
SACOL0247 lrgA holin-like protein LrgA Biosynthesis and degradation of murein sacculus and peptidoglycan −4.64
SACOL0052 NA glycosyl transferase, group 1 family protein Biosynthesis and degradation of surface polysaccharides and lipopolysaccharides −4.19
SACOL1195 mraY phospho-N-acetylmuramoyl-pentapeptide-transferase Biosynthesis and degradation of murein sacculus and peptidoglycan −3.71
SACOL0697 tagX teichoic acid biosynthesis protein X −3.51
SACOL0801 murB UDP-N-acetylenolpyruvoylglucosamine reductase −2.97
SACOL1023 murE UDP-N-acetylmuramoylalanyl-D-glutamate--2,6-diaminopimelate ligase −2.89
SACOL1194 pbp1 penicillin-binding protein 1 −2.5
SACOL1196 murD UDP-N-acetylmuramoylalanine--D-glutamate ligase −2.45
SACOL2074 NA D-alanine--D-alanine ligase −2.39
SACOL1687 NA N-acetylmuramoyl-L-alanine amidase, family 3 Biosynthesis and degradation of surface polysaccharides and lipopolysaccharides −2.38
SACOL0543 glmU UDP-N-acetylglucosamine pyrophosphorylase −2.21
SACOL2103 NA UDP-N-acetylglucosamine 2-epimerase −2.21
SACOL0033 mecA penicillin-binding protein 2 Biosynthesis and degradation of murein sacculus and peptidoglycan −2.15
SACOL1329 femC glutamine synthetase FemC −2.15
SACOL0242 NA teichoic acid biosynthesis protein, putative −2.12
SACOL1134 kdtB lipopolysaccharide core biosynthesis protein KdtB Biosynthesis and degradation of surface polysaccharides and lipopolysaccharides −2.09
SACOL1609 pbp3 penicillin-binding protein 3 Biosynthesis and degradation of murein sacculus and peptidoglycan −2.01
SACOL1453 murG UDP-N-acetylglucosamine--N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase −1.98
SACOL0263 lytM peptidoglycan hydrolase −1.96
SACOL0695 NA tagG protein, teichoic acid ABC transporter protein, putative −1.87
SACOL1066 NA fmt protein −1.83
SACOL1062 atl bifunctional autolysin Biosynthesis and degradation of surface polysaccharides and lipopolysaccharides −1.78
SACOL1424 NA phosphate ABC transporter, phosphate-binding protein Other −1.74
SACOL1790 murC UDP-N-acetylmuramate--alanine ligase Biosynthesis and degradation of murein sacculus and peptidoglycan −1.58
SACOL1951 NA Mur ligase family protein −1.57
Upregulated Genes
SACOL2571 cwrA conserved hypothetical protein Conserved 24.05
SACOL1434 NA alanine racemase family protein Biosynthesis and degradation of murein sacculus and peptidoglycan 21.26
SACOL0147 cap5L capsular polysaccharide biosynthesis protein Cap5L Biosynthesis and degradation of surface polysaccharides and lipopolysaccharides 4.22
SACOL0150 cap5O capsular polysaccharide biosynthesis protein Cap5O 4.02
SACOL0148 cap5M capsular polysaccharide biosynthesis galactosyltransferase Cap5M 3.98
SACOL0151 cap5P UDP-N-acetylglucosamine 2-epimerase Cap5P 3.61
SACOL0149 cap5N capsular polysaccharide biosynthesis protein Cap5N 3.55
SACOL1161 murI glutamate racemase Biosynthesis and degradation of murein sacculus and peptidoglycan 3.11
SACOL0146 cap5K capsular polysaccharide biosynthesis protein Cap5K Biosynthesis and degradation of surface polysaccharides and lipopolysaccharides 2.95
SACOL0144 cap5I capsular polysaccharide biosynthesis protein Cap5I 2.83
SACOL0145 cap5J capsular polysaccharide biosynthesis protein Cap5J 2.79
SACOL0140 cap5E capsular polysaccharide biosynthesis protein Cap5E 2.67
SACOL0141 cap5F capsular polysaccharide biosynthesis protein Cap5F 2.17
SACOL2092 murAA UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1 Biosynthesis and degradation of murein sacculus and peptidoglycan 2.1
SACOL0143 cap5H capsular polysaccharide biosynthesis protein Cap5H Biosynthesis and degradation of surface polysaccharides and lipopolysaccharides 2.04
SACOL0699 pbp4 penicillin-binding protein 4 Biosynthesis and degradation of murein sacculus and peptidoglycan 2.04
SACOL0142 cap5G UDP-N-acetylglucosamine 2-epimerase Cap5G Biosynthesis and degradation of surface polysaccharides and lipopolysaccharides 1.99
SACOL0136 cap5A capsular polysaccharide biosynthesis protein Cap5A 1.68
SACOL0137 cap5B capsular polysaccharide biosynthesis protein Cap5B 1.67
SACOL2116 murAB UDP-N-acetylglucosamine 1-carboxyvinyltransferase 2 Biosynthesis and degradation of murein sacculus and peptidoglycan 1.67
Cellular processes
Downregulated Genes
SACOL0935 dltA D-alanine-activating enzyme/D-alanine-D-alanyl carrier protein ligase Toxin production and resistance −11.93
SACOL2295 NA staphyloxanthin biosynthesis protein, putative Pathogenesis −8.55
SACOL1535 srrA DNA-binding response regulator SrrA −8
SACOL0244 scdA ScdA protein Cell division −6.24
SACOL0270 NA staphyloxanthin biosynthesis protein, putative Pathogenesis −5.55
SACOL0095 spa immunoglobulin G binding protein A precursor −5.54
SACOL1437 NA cold shock protein, CSD family Adaptations to atypical conditions −4.87
SACOL2126 luxS autoinducer-2 production protein LuxS Other −4.15
SACOL0956 kapB kinase-associated protein B Adaptations to atypical conditions −3.8
SACOL1193 NA cell division protein FtsL Cell division −3.6
SACOL0608 sdrC sdrC protein Cell adhesion −3.53
SACOL1328 glnR glutamine synthetase repressor Toxin production and resistance −3.35
SACOL2291 NA staphyloxanthin biosynthesis protein Pathogenesis −3.33
SACOL0552 NA general stress protein 13 Adaptations to atypical conditions −3.32
SACOL1534 srrB sensor histidine kinase SrrB Pathogenesis −3.27
SACOL0743 bacA bacitracin resistance protein Toxin production and resistance −3.23
SACOL1396 fmtC fmtC protein −3.18
SACOL2075 ftsW cell division protein, FtsW/RodA/SpoVE family Cell division −3.12
SACOL1197 divIB cell division protein −2.91
SACOL0766 saeR DNA-binding response regulator SaeR Pathogenesis −2.87
SACOL1205 NA cell-division initiation protein, putative Cell division −2.86
SACOL0452 ahpC alkyl hydroperoxide reductase, C subunit Detoxification −2.8
SACOL1537 scpB segregation and condensation protein B Cell division −2.72
SACOL1624 era GTP-binding protein Era −2.46
SACOL1538 scpA segregation and condensation protein A −2.31
SACOL1184 NA exfoliative toxin, putative Toxin production and resistance −2.14
SACOL0746 norR transcriptional regulator, MarR family −2.06
SACOL2731 NA cold shock protein, CSD family Adaptations to atypical conditions −2.06
SACOL1383 mscL large conductance mechanosensitive channel protein −1.96
SACOL2024 agrD accessory gene regulator protein D Pathogenesis −1.91
SACOL0610 sdrE sdrE protein Cell adhesion −1.89
SACOL1410 femA femA protein Toxin production and resistance −1.76
SACOL0765 saeS sensor histidine kinase SaeS Pathogenesis −1.69
SACOL0245 lytS sensor histidine kinase LytS −1.66
SACOL1198 ftsA cell division protein FtsA Cell division −1.53
Upregulated Genes
SACOL1943 vraS sensor histidine kinase VraS Toxin production and resistance 5.22
SACOL1450 arlS sensor histidine kinase ArlS Pathogenesis 4.74
SACOL2353 tcaR transcriptional regulator TcaR Toxin production and resistance 4.18
SACOL0672 sarA staphylococcal accessory regulator A 4.11
SACOL2157 NA drug resistance transporter, EmrB/QacA subfamily 3.75
SACOL1451 arlR DNA-binding response regulator ArlR Pathogenesis 3.61
SACOL2289 sarY staphylococcal accessory regulator Y Toxin production and resistance 3.21
SACOL2258 sarV staphylococcal accessory regulator V 2.87
SACOL1942 vraR DNA-binding response regulator VraR 2.61
SACOL1003 NA negative regulator of competence MecA, putative DNA transformation 2.01
SACOL0608 sdrC sdrC protein Cell adhesion 1.82
Protein fate
Downregulated Genes
SACOL1801 NA peptidase, M20/M25/M40 family Degradation proteins, peptides, and glycopeptides −3.45
SACOL0806 pepT peptidase T −2.89
SACOL2385 NA heat shock protein, Hsp20 family Protein folding and stabilization −2.77
SACOL1946 NA methionine aminopeptidase, type I Protein modification and repair −2.62
SACOL1777 NA serine protease HtrA, putative Degradation proteins, peptides, and glycopeptides −2.42
SACOL0581 secE preprotein translocase, SecE subunit Protein and peptide secretion and trafficking −2.13
SACOL1588 NA proline dipeptidase Degradation proteins, peptides, and glycopeptides −2.12
SACOL2038 NA metalloendopeptidase, putative, glycoprotease family −2.06
Upregulated Genes
SACOL0979 clpB ATP-dependent Clp protease, ATP-binding subunit ClpB Degradation proteins, peptides, and glycopeptides 9.42
SACOL1433 NA peptidase, M20/M25/M40 family 8.7
SACOL2125 NA peptidase, M20/M25/M40 family 7.13
SACOL0570 clpC ATP-dependent Clp protease, ATP-binding subunit ClpC, authentic frameshift 5.92
SACOL1636 dnaJ dnaJ protein Protein folding and stabilization 4.94
SACOL0833 clpP ATP-dependent Clp protease, proteolytic subunit ClpP Degradation proteins, peptides, and glycopeptides 3.72
SACOL1638 grpE heat shock protein GrpE Protein folding and stabilization 3.5
SACOL0968 spsA signal peptidase IA, inactive Protein and peptide secretion and trafficking 2.98
SACOL1637 dnaK dnaK protein Protein folding and stabilization 2.67
SACOL2438 NA endopeptidase, putative Degradation proteins, peptides, and glycopeptides 2.46
SACOL1459 NA peptide methionine sulfoxide reductase, degenerate Protein modification and repair 2.38
SACOL2016 groEL chaperonin, 60 kDa Protein folding and stabilization 2.36
SACOL0556 NA chaperonin, 33 kDa 2.17
SACOL0844 secG preprotein translocase, SecG subunit Protein and peptide secretion and trafficking 2.13
SACOL0969 spsB signal peptidase IB 2.02
SACOL2017 groES chaperonin, 10 kDa Protein folding and stabilization 1.81
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a

NA – Gene symbol not available

Related to the cell envelope, classified under Cellular Processes, approximately 54 and 49 genes were down- and up-regulated, 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, 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, staphylococcal accessory regulator sarA, sarV, sarY were upregulated between 2 to 5 fold (Table 2).

Another prominent category of genes altered by CPV treatment is the Protein Fate, set of genes which includes 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 degradation of proteins, peptides, and glycopeptides were down regulated between -4 to -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 to 5 fold (Table 2).

In addition to cell wall related genes, a variety of genes involved in DNA metabolism that play a role in DNA replication, recombination, and repair were down- and up-regulated by CPV challenge (Supplementary 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 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 ultra structure typical of S. 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 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 S. aureus.

Figure 2.

Figure 2

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Electron micrograph of ½ x MIC of CPV treated S. aureus COL cells. A, control; B, cells after 30 min of CPV treatment. Magnification, × 30,000, 1 mm = 33.33 nm.

DISCUSSION

The infections caused by MRSA and VISA due to 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 S. 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 S. aureus strain varied in the disc diffusion screening assay. The VISA strain 13136 p−m+ 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 by disc diffusion and agar dilution methods (Prabuseenivasan et al. 2006, Chao et al. 2008, Fisher and Phillips 2006, Dorman and Deans 2000, Burt and Reinders 2003, Abulrob et al. 2004, Nostro et al. 2004, Busatta 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, Cox et al. 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 sub-inhibitory concentration to obtain an in depth understanding of mode of action of antimicrobials (Wecke and Mascher 2011). Accordingly, in our study S. aureus genome microarrays were used to capture the genomic response of CPV treated S. 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 S. aureus cells during the CPV treatment supported the CPV induced cell wall damage in S. 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. 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 (Balibar et al. 2010). 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 Escherichia coli and S. 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 S. aureus is not specific on cytoplasmic membrane but due 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 S. 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 S. aureus cells and those genes are discussed in the following sections.

The PBPs are membrane-associated proteins that catalyze the final step of murein biosynthesis of cell-wall peptidoglycan in S. 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 S. 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 S. 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 S. 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 S. 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), teichoicacids, 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 S. 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 S. aureus cellular processes were affected by CPV treatment. D-alanine-activating enzyme/D-alanine-D-alanyl carrier protein ligase encoding dltA, dltB, dltC, dltD were reportedly down regulated in CPV treated cells. dltABCD operon controls the alanylation of wall teichoicacids which are involved in the control of autolysin activity in S. aureus. Previous research by Peschel et al. (1999; 2000) demonstrated that mutation in dlt genes lead to failure of alanylation in the teichoic acids and consequently S. aureus cells become sensitive to human defensin HNP1–3, animal-derived protegrins, tachyplesins, and magainin II, and to 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 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 as well as membranes of the S. aureus cells.

In S. 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 two component of these response regulators along with arlSR associated accessory regulators sarA, sarV, sarY in response to CPV treatment could be contemplated as a S. aureus protective response to the cell wall damage caused by the down regulation 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 S. aureus cell walls and caused profound cell wall damage and cell lysis. Treatment of S. 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 S. aureus increased the chaperones to restore the lysed proteins. Therefore, we speculate that the genes associated with 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 cell wall related genes, DNA-replication, -recombination, and -repair related genes were also apparently down- and up-regulated in CPV treated cells. In previous studies some of these genes have been identified as members of the cell wall stress stimulon. In S. 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 β-lactam antibiotics (Perez-Capilla et al. 2005). Interestingly, in our study approximately 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 S. 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 S. aureus cells exhibiting down regulation of cell envelope related genes and corroborated the cell wall active and lytic effect of CPV on S. 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 S. aureus cells. Similar cytoplasmic losses have also been reported in tea tree oil treated S. aureus cells (Carson et al. 2002). Apparently the down regulation 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 S. 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.

Supplementary Material

Supp Table S1

Table S1. S. aureus COL genes upregulated in response to CPV treatment

Supp Table S2

Table S2. S. aureus COL genes downregulated in response to CPV treatment

Acknowledgments

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).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp Table S1

Table S1. S. aureus COL genes upregulated in response to CPV treatment

NIHMS359571-supplement-Supp_Table_S1.xls (174.5KB, xls)
Supp Table S2

Table S2. S. aureus COL genes downregulated in response to CPV treatment

NIHMS359571-supplement-Supp_Table_S2.xls (221.5KB, xls)

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