Friday, 27 March 2015

Natural Plant Products Used against Methicillin-Resistant Staphylococcus aureus

Chapter 2 – Natural Plant Products Used against Methicillin-Resistant Staphylococcus aureus


Methicillin-resistant Staphylococcus aureus (MRSA) is currently one of the most common causes of serious infections, in both hospital and community environments. This type of resistance was discovered in the 1960s, and now (50 years later) it has not been possible to develop an effective therapy to fight against it. This chapter compiles the results of several studies demonstrating the activity (mainly in vitro) of various extracts, essential oils, and plant-derived molecules that can act alone or in combination against MRSA. In addition, natural alternatives (other than from plants) being studied for the same purpose are described. Finally, the important role of phytotherapy in the development of new therapies against multidrug-resistant bacteria such as MRSA is emphasized.


  • methicillin-resistant Staphylococcus aureus;
  • phytotherapy;
  • ethnopharmacology;
  • plant extracts;
  • bacterial drug resistance


Following its discovery, penicillin began to be used as a treatment for Staphylococcus aureus infections in the 1940s, initially providing very good results. However, just 1 year after the implementation of this antibiotic, penicillin-resistant strains were identified [1]. Such resistance is caused by the production of plasmid-encoded β-lactamase. Since the 1950s, there has been a substantial and continuous increase in penicillin resistance. The use of other antistaphylococcal drugs, such as chloramphenicol, erythromycin, streptomycin, sulfonamides, and tetracyclines, led to resistance to these drugs in S. aureus in the 1950s. The introduction of methicillin (a semisynthetic penicillin) in 1960 marked a major advance in the treatment of staphylococcal infections resistant to penicillin; however, the first strain of methicillin-resistant S. aureus (MRSA) was described in 1961, just a few months after onset of clinical use of the antibiotic [2]. The main resistance mechanism of MRSA is the production of a penicillin-binding protein (PBP) with lower affinity for β-lactam, called PBP2a. This resistance is encoded by the mecA gene and is part of a mobile element located within a pathogenicity island known as staphylococcal chromosomal cassette mec (SCCmec) [3]. MRSA is resistant to all β-lactams (including carbapenems and cephalosporins) [4] and usually resistant to aminoglycosides, clindamycin, erythromycin, quinolones, rifampin, sulfonamides, and tetracyclines, while it is usually susceptible to glycopeptides [5]. In 1996 in Japan, a strain of MRSA was isolated with reduced susceptibility to vancomycin [6], exhibiting a minimum inhibitory concentration (MIC) of 8 μg/mL. Subsequently, isolates with the same characteristics were reported in other countries. In 2002 in the United States, the first MRSA strain with high resistance was detected (MIC > 32 μg/mL) that had acquired a vanA gene identical to that of vancomycin-resistant enterococci [7].
MRSA infection often occurs endogenously in sites with previous disturbance of the mucocutaneous barrier due to traumatic injury, surgery, intravenous drug abuse, skin diseases, and intravascular catheterization, among others. This causes further colonization with MRSA, resulting in infections of the skin and subcutaneous tissue (abscesses), wound infections, and intravascular or urinary catheter-related infection. The spread of bacteria by blood can cause septic shock and severe metastatic infections such as acute endocarditis, arthritis, meningitis, myocarditis, pericarditis, pneumonia, and osteomyelitis [8].
As described above, bacteria such as S. aureus develop resistance mechanisms easily over a short time; this resistance rate is much higher than the rate of creation or discovery of new antibiotics, which makes it important to investigate therapeutic alternatives derived from various sources. Plants represent an important source of antibacterial molecules, some of which are known and probably others that are unknown. Consequently, a fourfold increase in the number of publications since the late 2000s compared with the first 5 years of the 1990s reflects the increased research interest in this field. In the next sections, some of the most important results of research studies aimed at combating multidrug resistance in S. aureus with plant-derived compounds are shown.

Plants Extracts with Anti-Methicillin-Resistant Staphylococcus aureus Effects

Antibiotic resistance of microorganisms is a growing problem in public health. It is considered as an emerging phenomenon worldwide that has led to the increased resistance of pathogenic microorganisms involved in the development of nosocomial and community acquired infections.
However, in relation to the total number of plant species in the world, the plants that have already been studied in order to evaluate their antimicrobial properties represent a tiny amount and a large number of scientists around the world are studying this phenomenon. This interest is mainly due to the development of bacterial strains with multiple resistance to antibiotics, a problem that is as old as the use of antibiotics itself. Thus, over time researchers have developed several techniques that enable the study of antimicrobial effects. In order to demonstrate an effect or activity of a plant against a living organism, it is necessary to separate the active principle(s) from the inert or inactive components. The most common way to do this is by exposing small amounts of plant tissues (fresh or dried) to water or organic solvent, depending on the nature of compound to be obtained (polar solvents for hydrophilic compounds and nonpolar for lipophilic compounds), or by successive extractions using different solvents to obtain different nature extracts. Another way is to obtain essential oil (EO) of a plant using techniques such as hydrodistillation [9].
Once the extracts or EOs are obtained, bioassays may be carried out. This involves exposing organisms to decreasing concentrations of extract or EO and determining the MIC. In this procedure, one should take into account that concentrations > 1 mg/mL (in the case of an extract) may produce false-positive results; therefore, candidates are those plants extracts that show antimicrobial activity at < 100 μg/mL [10]. Several methods that can be used to establish the MIC include agar dilution (plate dilution), microdilution (microtiter wells), macrodilution (in tubes), and indirectly by agar diffusion, among others. For these tests, the most commonly used culture medium is Mueller-Hinton in either agar or broth form (depending on the bioassay technique). This culture medium is accepted by the Clinical and Laboratory Standards Institute (CLSI) for evaluating the antimicrobial susceptibility of most bacterial strains. According to the selected technique, a specific bacterial concentration is used, but in general the standard reference is a concentration equivalent to tube number 0.5 in the McFarland scale: this represents a turbidity of 1.5 × 108 colony-forming units (CFU) per mL. This bacterial concentration is also used established to test antimicrobial susceptibility by the agar diffusion method. Results are frequently achieved after 18–24-h incubation at a temperature range of 35–37 °C (the temperatures at which most pathogenic bacteria thrive) [11].
The antimicrobial effect may be indicated by the MIC or the minimum bactericidal concentration (MBC), according to the technique used. Some researchers also report the IC50 (50% inhibitory concentration) or IC90 (90% inhibitory concentration) values. For extracts, the MIC is reported in units of mg/mL or smaller. For EOs, the MIC or MBC is usually expressed in units of μL/mL or % v/v. As mentioned above, several research groups are working on this issue worldwide. Table 2-1 lists some plants that have been investigated and shown to possess antimicrobial activities, either as an antibiotic adjuvant or by direct action against MRSA.
TABLE 2-1. Selected Plants Whose Extracts and Essential Oils Have Anti-Methicillin-Resistant Staphylococcus aureus Activities
PlantFamilyPlant partMICReference
Alchornea cordifoliaEuphorbiaceaeLeaves3.1 mg/mL[12]
Persea americanaLauraceaeLeaves6.3 mg/mL
Solanum verbascifoliumSolanaceaeLeaves6.3 mg/mL
Rosmarinus officinalisLamiaceaeLeaves0.13–3.13 mg/mL[13]
Thymus vulgarisLamiaceaeAerial parts18.5 μg/mL[14]
Eucalyptus globulusMyrtaceaeAerial parts85.6 μg/mL
Chaerophyllum libanoticumApiaceaeFruit0.25 mg/mL[15]
Zataria multifloraLamiaceaeAerial parts0.5–1 μL/mL (Eo)[16]
Atuna racemosaChrysobalanaceaeSeeds16–32 μg/mL[17]
Cleistocalyx operculatusMyrtaceaeBuds4–16 μL/mL (Ext)
8 mg/mL (Eo)
Punica granatumLythraceaeFruit, pericarp250 mg/L[19]
Tabebuia avellanedaeBignoniaceaeWood125 – > 250 mg/L
Dendrobenthamia capitataCornaceaeAerial parts1.25 ± 0.18 mg/mL[20]
Elsholtzia rugulosaLamiaceaeWhole plant1.43 ± 0.13 mg/mL
Elsholtzia blandaLamiaceaeAerial parts1.32 ± 0.16 mg/mL
Geranium strictipesGeraniaceaeRoot1.34 ± 0.30 mg/mL
Polygonum multiflorumPolygonaceaeRoot1.34 ± 0.22 mg/mL
Emblica officinalisEuphorbiaceaeSeedsSynergy with AMX[21]
Nymphaea odorataNymphaeaceaeStamenSynergy with AMX
Hypericum perforatumHypericaceaeAerial parts1 μg/mL[22]
Ecballium elateriumCucurbitaceaeFruit0.19–1.56 mg/mL[23]
Arthrocnemum indicumAmaranthaceaeBuds4–8 mg/mL[24]
Salicornia brachiataAmaranthaceaeBuds4–8 mg/mL
Suaeda monoicaChenopodiaceaeLeaves4–8 mg/mL
Suaeda maritimeChenopodiaceaeLeaves4–8 mg/mL
Sesuvium portulacastrumAizoaceaeLeaves4–8 mg/mL
Avicennia officinalisAcanthaceaeLeaves2–4 mg/mL
Ceriops decandraRhizophoraceaeLeaves1–2 mg/mL
Aegiceras corniculatumMyrsinaceaeLeaves0.5–1 mg/mL
Excoecaria agallochaEuphorbiaceaeLeaves0.1–0.2 mg/mL
Lumnitzera racemosaCombretaceaeLeaves0.5–1 mg/mL
Acanthus ilicifoliusAcanthaceaeLeaves2–4 mg/mL
Olea europaeaOleaceaeLeaves0.8–12.5% v/v (Eo)[25]
ApiaceaeFruit0.25 mg/mL (EO)[26]
Boswellia spp.BurseraceaeBark17.3–42.1 mg/mL (oleo-gum resin)[27]
Melaleuca alternifoliaMyrtaceaeLeaves0.04% v/v[28]
AMX, amoxicillin, Eo, essential oil; Ext, extract; MIC, minimal inhibitory concentration.
As shown in Table 2-1, various plant families have been shown to possess antimicrobial effect, indicating that their distribution is ubiquitous. This indicates that plants from all regions of the world may be natural sources of antimicrobials, which may therefore generate a wide field of research.
Furthermore, various different parts of the plants can be studied, ranging from the seed to the fruit, and even the entire plant. It is important to ensure that the plant resources used should be quickly renewable and that the maximum amount of material should be obtained from each plant for obtaining the extract or metabolite. The leaves are the parts of the plant that best fulfills these characteristics, because they are the most abundant elements of the plant and are quickly renewed after harvesting. Although leaves are the ideal source material, we should not ignore other parts of the plant, since both the distribution and concentration of metabolites can vary from one tissue to another.
Regarding the MIC of extract required to inhibit for MRSA, studies have reported concentrations ranging from 250 mg/mL to 1 μg/mL. However, it is important to remember that some authors believe that concentrations > 1 mg/mL (in extracts) are not significant, since the inhibitory effect may not relate to the activity of the antimicrobial or metabolite(s) but may instead be a result of other effects, such as osmotic pressure exerted by solutes on the bacterial cell walls [10].

Molecules with Anti-Methicillin-Resistant Staphylococcus aureus Activity Derived from Plants

Once a plant extract is found to have an antimicrobial effect, the next logical step is to determine which compounds or molecules are involved in this process. This can be accomplished using separation techniques such as high performance liquid chromatography (HPLC) or gas chromatography-coupled mass spectrometry, among others. The basis of the technique is the same, but some adjustments are made in order to work with extracts. Sometimes, after identifying the antimicrobial molecule, modifications are performed to the purified compound in an attempt to increase its antimicrobial activity [29].
The wide diversity and large number of molecules that may be contained in a natural extract raises the possibility that the constituent molecules may react to generate effects such as synergism or antagonism. Thus, some individual molecules may have stronger effects than the whole extract (i.e., there is an antagonist within the extract) or a reduced effect (i.e., there is a synergistic factor in the extract) [30]. However, Ríos et al. [10] propose that individual molecules that have MICs > 1 mg/mL should not be considered as inhibitors.
Table 2-2 lists some molecules with demonstrated antimicrobial effect against MRSA. It can be noted that the different MICs reported are almost all in the range of micrograms per milliliter (μg/mL), but concentrations expressed in micromoles (μM) are also reported. Micromoles is considered to be the most appropriate unit for assessing molecules (because of size variations), but it is confusing to compare micromoles with concentrations expressed in mg/mL (or similar) units. To avoid confusion, it is recommended to report molecular concentrations in terms of μM. Rescaling can be then done by dividing the amount in μg/mL by the molecular weight of the substance and multiplying this result by 1,000 to obtain μM or μmol/L, which is the same.
TABLE 2-2. Molecules Extracted from Plants that Have Anti-Methicillin-Resistant Staphylococcus aureus Activity
Rosmarinus officinalis/aerial partsLamiaceaeCarnosic acid32–64 μg/mL[31]
Carnosol16 μg/mL
4′,7-dimethoxy-5-hydroxy-flavone16–32 μg/mL
12-methoxy-trans-carnosic acid16–64 μg/mL
Hypericum japonicum/aerial partsHypericaceaetaxifolin-7-O-α-l-rhamnopyranoside8–64 μg/mL[32]
aromadendrin-7- O-α-l-rhamnopyranoside64–128 μg/mL
quercetin-7-O-α-l-rhamnopyranoside> 2048 μg/mL
Inula hupehensis/rootAsteraceae8-hydroxy-9,10-diisobutyloxythymol62.3 μg/mL[33]
Calophyllum thwaitesii/NRClusiaceaecalozeyloxanthone4.1–8.1 μg/mL[34]
Calophyllum moonii/NR6-deoxy-γ-mangostin0.25 mg/mL
Punica granatum/pericarpLythraceaeα-lapachone62.5 mg/L[35]
α-xyloidone125–250 mg/L
Tabebuia avellanedae/woodBignoniaceaeα -nor-lapachone15.6–31.2 mg/L
α -nor-hydroxylapachone15.6–62.5 mg/L
Sophora flavescens/rootsFabaceaeSophoraflavanone G2–4 μg/mL[36]
Kuraridin8–16 μg/mL
Dendrobenthamia capitata/aerial partsCornaceaeBetulinic acid62.5–125 mg/mL[37]
Angelica dahurica/rootsApiaceaeC17-polyacetylene falcarindiol8–32 μg/mL[38]
Hypericum perforatum L./aerial partsHypericaceaePhloroglucin1 μg/mL[39]
Momordica balsamina/aerial partsCucurbitaceaeBalsaminol A50 μM[40]
Balsaminol B25 μM
Balsaminagenin F100 μM
Balsaminoside A50 μM
Karavilagenin C200 μM
7β-methoxycucurbita-5,24-diene-3β,23(R)-diol25 μM
Psoroma species/NRPannariaceaePannarin8 μg/mL (MIC90)[41]
Scutellaria baicalensis/NRLamiaceaeBaicalein64–256 μg/mL[42]
Podocarpus totara/leavesPodocarpaceaeTotarol4 μg/mL[43]
Artemisia gilvescens/leavesAsteraceaeSecoguaianolide sesquiterpene stereoisomer1.95 μg/mL[44]
Ulmus davidiana var. japonica/NRUlmaceaeMansonone F0.39–3.13 μg/mL[45]
Sophora alopecuroides/NRFabaceaeAlopecurones A–C3.13–6.25 μg/mL[46]
Garcinia mangostana/pericarpClusiaceaeRubraxanthone0.31–1.25 μg/mL[47]
Myrtus communis/leavesMyrtaceaeMyrtucommulone A0.5–2 μg/mL[48]
MIC, minimal inhibitory concentration; NR, not reported.
Looking at TABLE 2-1 and TABLE 2-2, we can see that the entries in the former can be more extensively compared. This is because the technologies necessary to extract and purify molecules are expensive and require specialized personnel. Although this can restrict the development of new antibiotics from natural plant sources, it is the logical way to develop most studies.
Regarding the type of molecules that can be studied, these belong to different classes but are mainly flavonoids and terpenoids.

Synergy between Natural Products and Antibiotics Used for the Treatment of Methicillin-Resistant Staphylococcus aureus

Synergy is the effect of the combined action of two or more substances (in this case antimicrobials), characterized by having a greater effect than that resulting from the sum of the effects of the individual substances [49].
The synergistic effect occurs when different constituents of an extract interact to increase a specific antibacterial effect or when they act on different targets, thus increasing the possibility of affecting several vital processes in bacterial reproduction or metabolism. This effect is most striking when an antibiotic is combined with an agent that antagonizes bacterial resistance mechanisms (as in the case of β-lactamase) [50].
Herbal medicine has the particular feature of providing consumers with synergistic effects of metabolites contained within extracts and may even generate effects in different systems, thus contributing to patient healing by treating several different sets of symptoms. This diversity is also observed in the mechanisms of action that various phytochemicals have on the multidrug-resistant bacteria. Among them are:
Modification of the receptor or active site (enhancing affinity to the antibiotic);
Enzymatic degradation and modification of bacterial enzymes that degrade antibiotics;
Increased permeability of the outer membrane; and
Inhibition of the efflux pump systems [51].
Several studies have been carried out to combat multidrug resistance in MRSA and have demonstrated the synergism between some naturally occurring molecules and chemotherapeutics to which the bacteria have become resistant (Table 2-3).
TABLE 2-3. Molecules and Extracts that have Synergy With Antibiotics Against Methicillin-Resistant Staphylococcus Aureus
PlantPartType of phytochemicalAntibioticReference
Rosmarinus officinalisLeavesEthanol extractCefuroxime[13]
Aerial partsCarnosic acidErythromycin, tetracycline[31]
Hypericum japonicumAerial partstaxifolin-7-O-α-l-rhamnopyranosideAmpicillin, ceftazidime, levofloxacin[32]
Zataria multifloraAerial partsEssential oilVancomycin[16]
Sophora flavescensRootsSophoraflavanone GCiprofloxacin, erythromycin, gentamicin, fusidic acid[36]
KuraridinCiprofloxacin, erythromycin, gentamicin, kanamycin, fusidic acid, oxacillin
Artemisia herba-albaLeavesMethanol extractChloramphenicol, erythromycin, gentamicin, Penicillin G[52]
Achillea santolinaLeaves and Flowers
Emblica officinalisSeedEthanol extractAmoxicillin[21]
Nymphaea odorataStamen
Scutellaria baicalensisNRBaicaleinCiprofloxacin, oxacillin[42]
Ecballium elateriumFruitEthanol extractPenicillin G[23]
Psoroma spp.NRPannarinGentamicin[41]
Podocarpus totaraLeavesTotarolMethicillin[43]
Stephania tetrandraNRTetrandrineAmpicillin, azithromycin, cefazolin, levofloxacin[53]
Turnera ulmifoliaLeavesEthanol extractGentamicin, kanamycin[54]
Sophora alopecuroidesNRAlopecurones A–CErythromycin, gentamicin, methicillin[46]
Garcinia mangostanaPericarpRubraxanthoneVancomycin[47]
NR, no report.
As shown in this table, there are more individual molecules than extracts studied. This may be explained by the large number of possible interactions that each of the molecules present in the extract has (some of which may exhibit antagonistic effects that mask a possible synergistic effect).
Synergism has been reported in combination with various antibiotics to which MRSA has resistance, suggesting that these antibiotics combined with the studied molecules could be used in the treatment of infections caused by this pathogen.
The strength of the studies mentioned here varies according to whether the whole extract or active molecule was tested, the number of combinations with different antibiotics, the strain tested (reference or clinical isolate), and the use of a positive control, among other features. Regarding the use of a positive control, it is important to note that these are not always available for all strains of MRSA due to the mechanism of resistance varying for each antibiotic. A possible control that could be used for molecules with a potential synergistic effect is reserpine; this alkaloid acts on resistant bacteria by inhibiting efflux pumps [55] and its use should be considered when testing selected antibiotics and MRSA strains that specifically exhibit this type of resistance.

Other Natural Sources with Effects on Anti-Methicillin-Resistant Staphylococcus aureus

Although plant extracts and their active molecules are widely studied by research groups around the world, other groups study alternative methods to overcome bacteria resistant to multiple antibiotics. Nature has provided a variety of compounds with potential antimicrobial activity and these sources are currently being explored with good results. The following sections provide a brief description of some natural alternatives to plant products that can be used against MRSA.

Small Peptides

It is known that some peptides have antimicrobial effects; these are distributed in many living organisms throughout the planet. One peptide that has given good results both in vitro and in vivo is the omiganan pentahydrochloride (MBI-226), a cationic peptide composed of 12 amino acids that interacts with the cytoplasmic membrane of Gram-positive and Gram-negative bacteria, causing changes in its polarity with subsequent destruction of the bacteria [56]. This peptide is effective against MRSA at an MIC of ≤ 64 mg/mL [57].
Recently, some studies using peptides generated de novo have given good results; for example, in 2010 Lee and colleagues reported that a peptide of 11 residues had an MIC of 5 μg/mL against MRSA, which makes it more effective than omiganan in vitro [58].

Molecules Derived from Animals

Some chemically modified molecules produced by animals are utilized as antimicrobials; such is the case for chitosan (chitin polysaccharide derived by N-deacetylation), which has a synergistic effect with β-lactams (ampicillin, oxacillin, and penicillin). The anti-MRSA activities of two deacetylated forms (aminoethyl-chitosan 90 and 50) have been studied. Results of this study showed synergy between aminoethyl-chitosan 50 and the three antibiotics mentioned above; these synergistic combinations dramatically decreased the MICs of the antibiotics [59].


Bacteriocins are antimicrobial compounds derived from bacteria that are generated as a competitive mechanism (antagonism) against other bacteria. An example of this type of bacterium is Pseudoalteromonas phenolica O-BC30T, which produces a bacteriocin chemically defined as 2,2′,3-tribromobiphenyl-4,4′-dicarboxylic acid that inhibits clinical isolates of MRSA with MICs in the range of 1–4 μg/mL [60].
Lactobacilli are also known for their ability to produce such substances; L. acidophilus and L. casei were reported to generate lactic acid derivatives that act as bacteriocins, thus inhibiting the growth of MRSA by direct antagonism [61].


There is such a diversity of molecules with potential antimicrobial activity that even the letters of the genetic code (i.e., nucleotides) have been shown to possess antimicrobial functions. A study in mice with burns infected with MRSA demonstrated that interleukin-10 (Il-10) antisense oligodeoxynucleotides have the capacity to exert a protective role against this microorganism in situ by acting as a mediator of abscess formation, which limits the infection and makes it impossible for the bacteria to migrate to the blood and hence to produce sepsis [62].


Since nanotechnology was established as a field of study with various applications in science and technology, its use has been increasing dramatically and some years ago nanoparticles began to be studied as potential antimicrobial agents. Research into this application has been increasing and it now has great potential to become a viable alternative to traditional antimicrobials. For example, studies developed by Nanda and colleagues [63] tested the activity of silver (Ag) nanoparticles against MRSA, with favorable results.
Another report described the use of polyacrylate nanoparticles covalently bound to penicillin, which prevent β-lactamases from inactivating penicillin, thus maintaining the activity of this antibiotic [64].
As described in previous sections, research into different therapeutic alternatives to traditional antibiotics is being carried out using a range of different sources and technologies, which comprise an important advance in the fight against multidrug resistance exhibited by some bacterial pathogens such as MRSA.


As has been said throughout this chapter, multiple resistance to antibiotics is a growing public health problem; more specifically, MRSA represents a high risk of infection in both hospital and community environments.
Since the 1980s, the production of new antibiotics has been declining such that 25 years later it has been reduced by two-thirds [65], suggesting that we are currently underestimating the ability of bacteria to generate mechanisms of resistance. The versatility of these microorganisms puts us at a disadvantage; thus, research into new sources of antibiotics should be a priority worldwide.
Following great efforts, different lines of research are now consolidating to try to solve this problem, and it is evident that several plants contain compounds that either individually or combined with known antibiotics are effective against multidrug-resistant bacteria like MRSA.
There is a need to identify alternative therapies against bacterial resistance and it is evident that compounds with potential antimicrobial activities exist in Nature. Evidence for this is provided by the large number of phytochemicals (extracts and EOs) and isolated molecules with antimicrobial activity that act in many different ways to combat MRSA and other multidrug-resistant bacteria.
The diversity of globally distributed flora provides the investigation of natural extracts with an almost infinite range of possibilities. Nature will surely give us the necessary substrates, along with suitable tools and knowledge, to contribute to the fight against pathogens with multiple antibiotic resistance.


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