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. Author manuscript; available in PMC: 2009 Aug 13.
Published in final edited form as: J Ethnopharmacol. 2008 May 13;118(3):418–428. doi: 10.1016/j.jep.2008.05.005

Effects of extracts from Italian medicinal plants on planktonic growth, biofilm formation and adherence of methicillin-resistant Staphylococcus aureus

Cassandra L Quave 1,*, Lisa RW Plano 2, Traci Pantuso 1, Bradley C Bennett 1
PMCID: PMC2553885  NIHMSID: NIHMS65669  PMID: 18556162

Abstract

One-third of botanical remedies from southern Italy are used to treat skin and soft tissue infection (SSTI). Staphylococcus aureus, a common cause of SSTI, has generated increasing concern due to drug resistance. Many plants possess antimicrobial agents and provide effective remedies for SSTI. Our aim was to investigate plants from different ethnobotanical usage groups for inhibition of growth and biofilms in methicillin-resistant S. aureus (MRSA).

Three groups were assessed: plant remedies for SSTI, plant remedies not involving the skin, and plants with no ethnomedical application. We screened 168 extracts, representing 104 botanical species, for activity against MRSA (ATCC 33593). We employed broth dilution methods to determine the MIC after 18 hours growth using an optical density (OD600nm) reading. Anti-biofilm effects were assessed by growing biofilms for 40 hours, then fixing and staining with crystal violet. After washing, 10% Tween 80 was added and OD570nm readings were taken.

Extracts from 10 plants exhibited an IC50 ≤32 μg/ml for biofilm inhibition: Lonicera alpigena, Castanea sativa, Juglans regia, Ballota nigra, Rosmarinus officinalis, Leopoldia comosa, Malva sylvestris, Cyclamen hederifolium, Rosa canina, and Rubus ulmifolius. Limited bacteriostatic activity was evident. The anti-biofilm activity of medicinal plants was significantly greater than plants without any ethnomedical applications.

Keywords: Italy, medicinal plants, biofilms, methicillin-resistant Staphylococcus aureus

1. Introduction

The increase of microbial resistance to antibiotics threatens public health on a global scale as it reduces the effectiveness of treatments and increases morbidity, mortality, and health care costs (Coast et al., 1996). Evolution of highly resistant bacterial strains has compromised the use of newer generations of antibiotics (Levy, 2002). Although the active constituents may occur in lower concentrations, plant extracts may be a better source of antimicrobial compounds than synthetic drugs (Cox and Balick, 1994). The phenomenon of additive or synergistic effects is often crucial to bioactivity (Aqil et al., 2006a; b; Kamatou et al., 2006) in plant extracts and in some cases, the activity is lost in purified fractions (Cos et al., 2006). Development of bacterial resistance to synergistic drug combinations, such as those found in plants, may be slower than for single drug therapies.

Only a small percent of plants have been investigated for their bioactivity (Cox and Balick, 1994). Furthermore, most studies on the antimicrobial activity of plant extracts have been restricted to analysis of their bacteriostatic and bactericidal properties. New assays investigating other potential roles, such as the mediation of pathogenicity via quorum sensing inhibition, have recently emerged (Rasmussen and Givskov, 2006). Investigations into the anti-pathogenic potential of natural products may open new avenues for drug development in the control of antibiotic resistant pathogens.

The use of plants against skin disease is a common practice in the popular medicine of most cultures, although the precise causation of disease and mechanism of cure is not always understood (Grierson and Afolayan, 1999; Srinivasan et al., 2001). While 1–3% of pharmaceutical drugs are used to treat skin disorders and wounds, 33% of botanical therapies in non-Western societies are used to this end (Mantle and Gok, 2001). The topical application of many plant remedies for the treatment of various skin conditions has proven effective due to the antimicrobial and anti-inflammatory properties of plant compounds.

In the Vulture-Alto Bradano area of Basilicata Province, southern Italy (Figure 1), wild plants are regularly gathered as sources of food and medicine, making up an integral facet of home healthcare (Pieroni et al., 2002b; Pieroni et al., 2002c; Pieroni and Quave, 2005; Quave et al., 2008). At least one-third of all medicinal plants utilized in the folk-pharmacopoeia of this area are applied towards the treatment of skin and soft tissue infection (SSTI) (Quave et al., 2008). Vulture-Alto Bradano is characterized by small scale agriculture and its inhabitants maintain strong ties with the land. The demographic and socioeconomic aspects of this area are described in previous works (Pieroni et al., 2002c; Quave and Pieroni, 2005; Quave et al., 2008).

Figure 1.

Figure 1

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Map of the study area: Vulture-Alto Bradano, Basilicata Province, Italy.

Staphylococcus aureus, a gram-positive species, is a ubiquitous colonizer of the skin and mucous membranes of humans and animals (Fauci et al., 1998). It is the most prominent etiological agent for SSTI and is a common target for natural product drug screenings. Many strains of S. aureus carry resistance genes for penicillin antibiotics, tetracyclines, methicillin and now, vancomycin. Methicillin-resistant S. aureus (MRSA) presents a significant threat to public health in the USA. A surveillance study on MRSA found that an estimated 94,360 patients in the USA had invasive MRSA infections in 2005, resulting in an estimated 18,650 deaths (Klevens et al., 2007), surpassing the mortality estimates for AIDS in the USA.

Biofilm-related infections caused by staphylococci are among the leading causes of nosocomial infection in the USA (Kong et al., 2006). Biofilms comprise sessile microbial communities that embed themselves within a self-made extracellular polymeric matrix. This phenotype confers some protection to bacteria against host defenses and impedes the delivery of certain large molecule antimicrobials (Lewis, 2001). Moreover, bacteria associated with biofilms grow slowly, and this restricts the efficacy of many antibiotic classes, including the β-lactams (Lewis, 2005). The first stages of staphylococcal biofilm formation involve adherence of planktonic bacterial cells to cellular or prosthetic surfaces, such as surgically implanted medical devices, including catheters, plates, screws, artificial joints and cardiac valves (Costerton et al., 1999). Biofilms present significant therapeutic barriers for many antibiotics and the discovery of agents which could prevent their formation or adherence would be of great use.

The objectives of this study were to evaluate extracts of south Italian plants for effects on growth, biofilm formation and adherence in MRSA. Our hypothesis was that plants used specifically for the treatment of SSTI would demonstrate higher mean activity for growth and biofilm inhibition than plant remedies not involving the skin or plants with no ethnomedical application.

2. Materials and Methods

2.1 Plant material and extraction

Plants from three ethnobotanical usage categories were selected for analysis: Group 1: medicinal plants for skin and soft tissue infection (SSTI) (n=25), Group 2: medicinal plants not involved in skin therapies (n=28), and Group 3: plants with no ethnomedical application (n=51). Plants were categorized based on previous ethnobotanical surveys conducted in the Vulture-Alto Bradano area of southern Italy (Pieroni et al., 2002b; Pieroni et al., 2002c; Pieroni and Quave, 2005; Pieroni and Quave, 2006; Quave et al., 2008). Parameters for collecting a sample of non-medicinal species of Vulture-Alto Bradano were that they grew in abundance and were flowering at the time of collection. Plants were field-collected in the Vulture-Alto Bradano area of southern Italy and identified using the standard botanic work Flora d’Italia (Pignatti, 2002). Familial nomenclature follows the current Angiosperm Phylogeny Group (Stevens, 2001 onwards). Bulk samples were separated by part (e.g., leaves, stems, flowers, and roots), cut into small pieces and dried in a plant drier using a heat and air flow source (35°C) for 48–72 h. In the case of herbaceous species, efforts were made to collect all parts of the plant, including both above-ground and below-ground parts. Only the above-ground parts were typically collected from trees and large shrubs. Bulk specimens were composed only of the targeted plant species – parts from other plants and soil were removed prior to drying and vacuum storage. Cross-contamination was avoided by using clean drying nets and storage containers. After drying, plant materials were packed into plastic bags with several silica packets and vacuum sealed to prevent mold growth. Voucher specimens were deposited at the Herbarium Lucanum (HLUC) in Potenza, Italy and Fairchild Tropical Botanic Gardens (FTG) in Miami, FL, USA. Bulk specimens were shipped to the US under USDA permit #DP63438 for bioassays.

Dry plant materials were ground into a fine powder using a homogenizer. Ethanolic extracts of all plant samples were made by soaking in 95% denatured EtOH using a ratio of 1g (plant material):10 ml (EtOH) for 72 h. Flasks were agitated daily. Water extracts were made by boiling 1g (plant material): 50 ml (dH2O) for 30 minutes. Extracts were vacuum-filtered and rotary-evaporated, then frozen and lyophilized. Stock concentrations of 10 mg/ml of dry extract in the excipient (DMSO or dH2O) were prepared, sterile filtered (0.2 μm) and stored in the dark at 4°C. The excipient (DMSO or dH2O) made up less than 5.1% of the final test solution for MIC assays and less than 2.5% for biofilm assays.

2.2 Bacteria and culture conditions

Methicillin-resistant Staphylococcus aureus (ATCC 33593) was grown in Tryptic Soy Broth (TSB) or on agar plates for 18 h at 37°C. A 0.5 McFarland Standard was used to create inoculum densities of 1.5 × 108 cfu/ml in PBS using the direct suspension method (Isenberg, 2004) for MIC and biofilm assays.

2.3 Determination of minimum inhibitory concentrations (MICs)

MICs were determined by the microtiter broth method (Amsterdam, 1996) in sterile flat-bottom 96-well polystyrene plates. Serial dilution techniques were used to determine the MIC50 and MIC90 of extracts at concentrations of 8–512 μg/ml after 18 h growth. Negative controls (cells + TSB), positive controls (cells + TSB + antibiotics – vancomycin, ampicillin, and trimethroprim-sulfamethoxazole), vehicle controls (cells + TSB + DMSO), and media controls (TSB) were included. Positive controls for antibiotics were prepared at 8–512 μg/ml via serial dilution techniques. All tests were performed in triplicate. Optical density readings were taken using a KC4 microplate reader at 600 nm at 0 and 18 hours post-inoculation. Results are reported as the MIC for growth at 18 hours post-inoculation. To account for the effect of extract color on the OD600nm reading, a formula for calculating percent inhibition was used. The mean % inhibition of replicate tests was used to determine the final MIC values.

%inhibition=(1−(ODt18−ODt0ODgc18−ODgc0))x100

ODt18 = optical density (600 nm) of the test well at 18 hours post-inoculation

ODt0 = optical density (600 nm) of the test well at 0 hours post-inoculation

ODgc18 = optical density (600 nm) of the growth control well at 18 hours post-inoculation

ODgc0 = optical density (600 nm) of the growth control well at 0 hours post-inoculation

2.4 Biofilm formation and adherence

A modified microtiter dish system (Christensen et al., 1985; Schadow et al., 1988; Yarwood et al., 2004) was employed to test the effect of plant extracts on biofilm formation and adherence. Assays were performed in sterile flat-bottom 96-well polystyrene plates. Serial dilution techniques were employed to determine the IC50 of extracts at sub-inhibitory (for growth) concentrations of 4–128 μg/ml after 40 h growth. Negative controls (cells + TSB), positive controls (cells + TSB + antibiotics – vancomycin, ampicillin, and trimethroprim-sulfamethoxazole), vehicle controls (cells + TSB + DMSO), and media controls (TSB) were included. Positive controls for antibiotics were prepared at 4–128 μg/ml via serial dilution techniques. All tests were performed in triplicate.

To test for the effects of extracts on biofilm formation, the appropriate concentration of extract was added to the test wells prior to inoculation. Plates were placed in a 37°C incubator and bacteria were shaken for 40 h. The contents of the wells were then aspirated, rinsed 3 times with PBS, and fixed by drying for 1 h in the 37°C incubator. Once the wells were fully dry, we added 200 μl of 0.1% crystal violet stain to wells to stain for 15 m. The excess stain was rinsed off with tap water and 200 μl of 10% Tween 80 was added to wells. We pulled off the stain adhering to the biofilm biomass with the Tween 80 and transferred to new 96-well plates for spectrophotometric analysis (OD570nm).

To test for the effects of extracts on biofilm adherence, biofilms were established in the 96-well plates by growing shaking for 20 h at 37°C. At 20 h post-inoculation, planktonic cells and TSB were aspirated and fresh TSB was added with the appropriate concentration of test extract. Plates were then placed back into the 37°C incubator on a shaker for 20 h. The staining methods were the same as those described above.

Replicate absorbance readings for each concentration tested were averaged and the average of the media control (TSB + extract) was subtracted. This value was then divided by the mean absorbance of the vehicle control and multiplied by 100. The concentration at which the extract depleted the biofilm biomass by at least 50% was labeled as the IC50.

2.5 Statistical analyses

The mean values and standard deviations of all replicates described in the above tests were calculated using Excel software. Differences between means were analyzed with a One-Way ANOVA, followed by multiple comparison analysis using the Bonferroni method on SPLUS software. Differences were considered significant with p-values < 0.05.

3. Results

3.1 Effects on planktonic growth of MRSA

Plant extracts demonstrated limited bacteriostatic activity (Table 1). Roughly 18% of the extracts tested demonstrated an IC50 of 256 or 512 μg/ml. Of those demonstrating activity, the majority were not associated with any particular ethnobotanical application. The mean percent inhibition of growth for each ethnobotanical usage group when screened at 512 μg/ml ranged from 15.93–19.46% (Group 1=19.46%, Group 2=15.93%, and Group 3=18.43%). Based on results from a One-Way ANOVA (p>0.05), we found that there was no significant difference in the mean bacteriostatic activity of extracts from plants in each ethnobotanical usage group (Table 2a).

Table 1.

Effect of plant extracts on growth and biofilm formation in MRSA (ATCC 33593). The MIC50 for growth was tested in the range of 8–512 μg/ml, whereas the IC50 for biofilm formation was tested from 4–128 μg/ml due to issues with the percent excipient (DMSO) in the test solution. A dash (−) represents that no IC50 was identified within the concentration range tested.

Family Botanic Name Voucher ID Plant Part Ethno-botanical Use* Extract Solvent ** MIC50 (Growth) ** IC50 (Biofilm Formation)
Adoxaceae Sambucus ebulus L. CQ-168 inflorescence N EtOH - -
leaves S EtOH - -
stems N EtOH - -
Sambucus nigra L. CQ-151 woody parts R EtOH - -
leaves S EtOH 512 -
dH2O - -
inflorescence S; R EtOH - -
dH2O - -
infructescence F EtOH - -
Alliaceae Allium cepa L. CQ-206 leaves; bulbs; roots S; M; F EtOH - -
Apiaceae Daucus carota L. CQ-215 leaves; stems N EtOH - -
inflorescence; infructescence N EtOH - -
Foeniculum vulgare ssp. piperitum (Ucria) Coutinho CQ-192 leaves; stems M; F EtOH - -
Foeniculum vulgare ssp. vulgare Mill. CQ-196 leaves; stems M EtOH - -
Tordylium apulum L. CQ-101 flowers; leaves; roots; stems N EtOH - -
Apocynaceae Vinca major L. CQ-117 flowers; leaves; roots; stems M EtOH - -
Aracaeae Arum italicum Mill. CQ-175 stems N EtOH - -
fruits N EtOH - -
stalks N EtOH - -
leaves S EtOH - -
Asphodelaceae Asphodelus microcarpus Salzm. & Viv. CQ-109 inflorescence N EtOH - -
leaves N EtOH 512 -
Asteraceae Achillea ageratum L. CQ-219 leaves; stems; flowers M EtOH 256 -
Achillea millefolium L. CQ-176 inflorescence M EtOH - -
leaves; stems M EtOH 512 -
leaves; stems; flowers M EtOH - -
Anacyclus tomentosus DC. CQ-167 leaves; stems; flowers N EtOH - -
Cichorium intybus L. CQ-106 basal leaves; roots F EtOH 512 -
dH2O - -
leaves; stems; flowers F EtOH - -
Matricaria recutita L. CQ-118 flowers; leaves; roots; stems S; M EtOH 512 -
dH2O - -
Scolymus hispanicus L. CQ-199 leaves; stems; flowers N EtOH - -
Tussilago farfara L. CQ-202 leaves; stems; roots S EtOH - -
Urospermum dalechampii (L.) Scop. CQ-134 flowers; leaves; roots; stems N EtOH - -
Boraginaceae Anchusa officinalis L. (CQ-128) CQ-128 leaves; stems; flowers N EtOH - -
Borago officinalis L. CQ-100 flowers; leaves; roots; stems M EtOH - -
dH2O - -
Cerinthe major L. CQ-110 flowers; leaves; roots; stems N EtOH - -
Echium italicum L. CQ-162 leaves; stems; flowers N EtOH - -
Brassicaceae Brassica rapa subsp. rapa CQ-104 flowers; leaves; roots; stems F EtOH - -
Cardaria draba (L.) Desv. CQ-140 flowers; leaves; roots; stems N EtOH 512 -
Eruca sativa Mill. CQ-102 flowers; leaves; roots; stems N EtOH - -
Sisymbrium officinale (L.) Scop. CQ-131 flowers; leaves; roots; stems N EtOH - -
Caprifoliaceae Lonicera alpigena L. CQ-213 woody parts N EtOH - 32
leaves N EtOH - -
Caryophyllaceae Saponaria officinalis L. CQ-210 leaves; stems; flowers N EtOH - -
Silene alba (Mill.) E.H.L. Krause CQ-123 leaves; stems; flowers N EtOH - -
Silene nutans L. CQ-125 leaves; stems; flowers N EtOH - -
Cucurbitaceae Ecballium elaterium (L.) A. Richard CQ-169 leaves; stems; flowers S EtOH 512 -
Dennstaedtiaceae Pteridium aquilinium (L.) Kuhn CQ-211 leaves N EtOH - -
stems N EtOH - -
Dipsacaceae Dipsacus fullonum L. CQ-201 leaves; stems N EtOH - -
flowers N EtOH - -
Knautia arvensis Coult. CQ-190 leaves; stems; flowers N EtOH - -
Knautia lucana Lacaita & Szabo CQ-166 leaves; stems; flowers N EtOH - -
Equisetaceae Equisetum arvense L. CQ-226 stems; leaves N EtOH - -
Fabaceae Acacia dealbata Link CQ-115 inflorescence O EtOH - -
stems O EtOH - -
leaves; stems O EtOH - -
Anthyllis vulneraria L. CQ-147 leaves; stems; flowers N EtOH - -
Astragalus monspessulanus L. CQ-112 leaves; stems; flowers; roots N EtOH - -
Coronilla emerus L. CQ-137 leaves; flowers N EtOH - -
woody stems N EtOH - -
Melilotus alba Medik. CQ-193 leaves; stems; flowers N EtOH 512 -
Robinia pseudoacacia L. CQ-155 woody parts N EtOH - -
leaves N EtOH 512 -
inflorescence N EtOH - -
Spartium junceum L. CQ-144 leaves; stems; flowers A EtOH - -
Trifolium repens L. CQ-138 leaves; stems; flowers; roots N EtOH - -
Vicia craca L. CQ-149 leaves; stems; flowers; roots N EtOH 512 -
Vicia faba L. CQ-103 leaves; stems; flowers; roots F EtOH - -
Vicia sativa subsp. angustifolio CQ-124 leaves; stems; flowers N EtOH 512 -
Vicia sativa subsp. sativa CQ-119 leaves; stems; flowers N EtOH - -
Wisteria sinensis (Sims) Sweet CQ-126 inflorescence O EtOH 512 -
stems O EtOH - -
leaves O EtOH 512 -
Fagaceae Castanea sativa Mill. CQ-191 inflorescence N EtOH 256 16
leaves N EtOH 256 -
woody parts A EtOH 512 -
Quercus cerris L. CQ-228 leaves N EtOH - -
stems; fruits N EtOH - -
Gentianaceae Centaurium pulchellum (Sw.) Druce CQ-217 leaves; stems; flowers; roots N EtOH - -
Geraniaceae Erodium ciconium (L.) L’Hér. CQ-142 leaves; stems; flowers; roots N EtOH - -
Erodium malacoides (L.) L’Hér. ex Aiton CQ-121 leaves; stems; flowers N EtOH 128 -
Geranium columbinum L. CQ-129 leaves; stems; flowers N EtOH 512 -
Hyacinthaceae Leopoldia comosa (L.) Parl. CQ-105 bulbs M; F EtOH - 16
dH2O - 8
leaves; inflorescence N EtOH - -
Hypericaceae Hypericum perforatum L. CQ-183 leaves; stems; flowers S EtOH 256 128
Juglandaceae Juglans regia L. CQ-181 immature fruits S; C EtOH - 16
leaves R EtOH - -
woody parts N EtOH 512 -
Juncaceae Juncus articulatus L. CQ-216 leaves; fruits N EtOH - -
Lamiaceae Ballota nigra L. CQ-160 stems S; M EtOH - -
roots N EtOH - -
leaves S; M EtOH - -
leaves; stems; flowers S; M EtOH - -
dH2O - 8
Clinopodium vulgare L. CQ-182 leaves; stems; flowers N EtOH - -
Marrubium vulgare L. CQ-170 leaves; stems; flowers S; M EtOH - -
dH2O - -
roots N EtOH - -
Mentha pulegium L. CQ-200 leaves; stems; flowers; roots F EtOH - -
Mentha spicata L. CQ-224 leaves; stems; flowers F EtOH - -
Origanum heracleoticum L. CQ-207 leaves; stems; flowers F EtOH - -
Phlomis herba-venti L. CQ-168 leaves; stems; flowers N EtOH - -
Rosmarinus officinalis L. CQ-113 leaves; stems; flowers F; S EtOH 512 16
Salvia pratensis L. CQ-165 leaves; stems N EtOH 512 -
Salvia virgata Jacq. CQ-127 leaves; stems; flowers N EtOH - -
Stachys tymphaea Hausskn. CQ-189 leaves; stems; flowers N EtOH - -
Liliaceae Lilium candidum L. CQ-174 leaves; stems N EtOH - -
inflorescence N EtOH - -
Malvaceae Alcea rosea L. CQ-205 leaves; stems; flowers; roots O EtOH - -
Malva sylvestris L. CQ-156 stems S; M EtOH - -
dH2O - 32
flowers S; M EtOH - -
leaves S; M EtOH 256 -
dH2O - 32
Moraceae Ficus carica L. CQ-173 leaves N EtOH - -
woody parts N EtOH - -
immature fruits S; F EtOH - -
Myrsinaceae Cyclamen hederifolium Aiton CQ-186 tubers M EtOH - 8
Nyctaginaceae Mirabilis jalapa L. CQ-222 leaves; flowers; fruits N EtOH - -
Oleaceae Olea europaea L. CQ-197 leaves N EtOH - -
woody parts A EtOH - -
Orchidaceae Aceras anthropophora R. Br. CQ-153 leaves; stems; flowers; roots N EtOH - -
Orchis italica Poir. CQ-133 inflorescence; leaves; stems N EtOH - -
Orchis purpurea Huds. CQ-132 inflorescence; leaves; stems N EtOH - -
Papaveraceae Fumaria officinalis L. CQ-107 leaves; stems; flowers; roots N EtOH - -
Papaver rhoeas subsp. rhoeas CQ-145 leaves; stems; flowers; roots F EtOH - -
Papaver somniferum L. CQ-178 leaves; stems; flowers; roots M; R EtOH - -
Plantaginaceae Digitalis ferruginea L. CQ-227 leaves; stems; flowers N EtOH - -
Linaria vulgaris Hill CQ-223 leaves; stems; flowers; roots N EtOH - -
Plantago major L. CQ-225 leaves; stems; flowers; roots S; M EtOH - -
Poaceae Agropyron repens (L.) P. Beauv. CQ-208 leaves; stems; roots M EtOH - -
Arundo donax L. CQ-146 stem internodes A; R EtOH - -
stem nodes S EtOH - -
dH2O - 128
leaves; stems A; R EtOH - -
Polygonaceae Rumex crispus L. CQ-171 leaves; stems; fruits S EtOH 512 -
Pottiaceae Syntrichia ruralis (Hedw.) Web. & Mohr CQ-229 whole plant N EtOH - -
Ranunculaceae Delphinium fissum Waldst. & Kit. CQ-187 leaves; stems; flowers; fruits N EtOH - -
Ranunculus acris L. CQ-135 leaves; stems; flowers N EtOH - -
Rosaceae Crataegus monogyna Jacq. CQ-116 leaves; stems; flowers M EtOH - -
Prunus spinosa L. CQ-163 woody parts; leaves M EtOH - -
fruits N EtOH 512 -
Rosa canina var. canina CQ-152 fruits N EtOH - 32
woody parts N EtOH - -
leaves; stems N EtOH 512 -
Rubus ulmifolius Schott CQ-164 leaves; stems; flowers S EtOH - -
leaves S EtOH - -
roots M EtOH 512 8
woody stems N EtOH 512 -
Rubiaceae Galium verum L. CQ-177 leaves; stems; flowers N EtOH - -
Scrophulariaceae Verbascum sinuatum L. CQ-218 leaves; stems; flowers N EtOH - -
Verbascum thapsus L. CQ-172 stems M EtOH - -
leaves M EtOH - -
inflorescence M EtOH - -
Ulmaceae Ulmus minor L. CQ-195 leaves N EtOH - -
woody parts M EtOH - -
Urticaceae Parietaria diffusa Mert. & Koch CQ-212 leaves; stems; fruits; roots M EtOH - -
Urtica dioica L. CQ-179 leaves; stems; flowers S; M; F EtOH - -
Valerianaceae Centranthus ruber (L.) DC. CQ-143 leaves; stems; inflorescence M EtOH 256 -
Vitaceae Vitis vinifera var. aglianico CQ-209 wine S; F - 64
stems N EtOH - -
fruits F EtOH - -
leaves N EtOH - -
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*

Ethnobotanical use of specific plant part(s) in the study region: S = medicinal application to skin; M = medicinal application not involving the skin; C = cosmetic applications; A = agricultural tool; O = ornamental; R = ritual or spiritual use; F = food; N = no reported use.

**

MIC50 and IC50 values expressed as μg/ml.

Table 2.

Statistical tests for differences in the inhibitory activity of the three ethnobotanical usage groups: Group 1 includes plants used for SSTI (n=25); Group 2 includes plants used medicinally, but not on the skin (n=28); and Group includes plants with no ethnomedical application (n=51). (a) One-way ANOVA tests were performed. These results indicate that there was no significant difference in the inhibition of planktonic growth (p > 0.05) between ethnobotanical usage groups. There was, however, a significant difference in the inhibition of biofilm formation (p< 0.05) between groups. (b) Multiple comparison analysis using the Bonferroni method was performed on the data for anti-biofilm activity. A significant difference in activity of Groups 1 and 3 is evident.

Df Sum of Sq Mean Sq F Value P Value
Planktonic Growth 2 960.1 480.0347 0.7320253 0.48121
Biofilm formation 2 2073.34 1036.669 7.159144 0.00104
(a)
Groups Compared Estimate Standard Error Lower Bound Upper Bound
1–2 2.13 3.25 −5.730 9.98
1–3 **** 8.15 2.36 2.460 13.90
2–3 6.03 2.75 −0.634 12.70
(b)
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3.2 Effects on MRSA biofilm formation

Extracts from 10 plants exhibited an IC50 ≤32 μg/ml for inhibition of biofilm formation: Lonicera alpigena, Castanea sativa, Juglans regia, Ballota nigra, Rosmarinus officinalis, Leopoldia comosa, Malva sylvestris, Cyclamen hederifolium, Rosa canina var. canina, and Rubus ulmifolius (Table 1). The mean percent inhibition of biofilm formation for each ethnobotanical usage group when screened at 64 μg/ml ranged from 0.58–8.73% (Group 1=8.73%, Group 2=6.6%, and Group 3=0.58%). Plants used in folk remedies for SSTI made up 38% of plants exhibiting an IC50 of 8–128 μg/ml for MRSA biofilm formation.

Base on the results from a One-Way ANOVA test (p<0.05), the mean anti-biofilm activity of extracts from plants in each ethnobotanical usage group was not the same (Table 2a). Multiple comparison analysis using the Bonferroni method revealed a significant difference between groups 1 and 3. The anti-biofilm activity of plants categorized for medicinal applications to the skin was significantly greater than activity from plants without any ethnomedical applications (Table 2b).

4. Discussion

A significant dose-dependent response in biofilm inhibition was evident in 5 of the 168 extracts tested: Arundo donax, Ballota nigra, Juglans regia, Leopoldia comosa, Marrubium vulgare, and Rubus ulmifolius (Figure 2). Here, we offer a discussion of the ethnomedicinal applications of these plants in southern Italy and review the literature for other reports of anti-staphylococcal activity.

Figure 2.

Figure 2

Figure 2

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The percent inhibition of extracts for planktonic growth, biofilm formation and adherence in MRSA. (a) Arundo donax, aqueous extract of nodes; (b) Ballota nigra, aqueous extract of aerial parts; (c) Juglans regia, ethanolic extract of immature fruits; (d) Leopoldia comosa, ethanolic extract of bulbs; (e) Marrubium vulgare, ethanolic extract of roots.

4.1 Arundo donax (Poaceae) - canna

The giant reed is commonly used in the construction of musical instruments and the production of industrial cellulose. It is also used for several other purposes in southern Italy, especially to support grape vines. This use was so important to local agriculture in the past that land was often divided along the border lines of reed colonies. The white hemicellulose membrane located at the nodes of the reed is inserted into fresh lacerations as a haemostatic agent (Passalacqua et al., 2007; Pieroni et al., 2002c) (Figure 3). Small tooth-sized pieces of the reed are also cut up and used in the ritual treatment of toothache (Quave and Pieroni, 2005). Antiproliferative activity against several human cancer cell lines has been reported for a lectin isolated from the rhizomes of this plant (Kaur et al., 2005). A. donax is rich in alkaloids, including bufotenidine and gramine. Small amounts of DMT have also been isolated from the flowers (Duke, 2008).

Figure 3.

Figure 3

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The white hemicellulose membrane found at the node of the giant reed (Arundo donax L. [Poaceae]) is used as a haemostatic agent for minor lacerations.

We found that aqueous extracts of the reed nodes (which contain the white hemicellulose membrane) demonstrated a marked dose-dependent response for anti-biofilm activity, both in preventing MRSA biofilm formation and disrupting established biofilms (Figure 2a). These results may suggest that the traditional application of the reed membrane to fresh lacerations may be useful as a prophylactic for biofilm-related infection. The effect of giant reed extract on biofilm formation and adherence in MRSA has not been previously reported.

4.2 Ballota nigra (Lamiaceae) – erba cane

Infusions of the aerial parts of black horehound are used in the south Italian pharmacopoeia as a rinse for skin rashes and are also drunk to promote circulation. Extensive phytochemical studies (Bertrand et al., 2000; Bruno et al., 1986; Seidel et al., 1997; Seidel et al., 2000) have been performed on black horehound, and several phenylpropanoid glycosides have been identified and linked to moderate growth inhibition in Staphylococcus aureus (Didry et al., 1999). Black horehound also contains diterpenes, such as marubiin, as well as caffeic and ferulic acid derivatives (Gruenwald et al., 1998). Potent antioxidant activity has reported in infusions of the aerial parts (Citoglu et al., 2004; Vrchovska et al., 2007). We prepared several types of extractions of the different parts of the plant, and found that an aqueous extraction of the aerial parts, similar to that prepared in the folk medical tradition, was the most effective at inhibiting both biofilm growth and adherence. Although this extract actually promoted planktonic growth of this MRSA strain by almost twofold, significant dose-dependent responses for the inhibition of both biofilm formation and adherence were evident (Figure 2b). The effect of black horehound extract on biofilm formation and adherence in MRSA has not been previously reported.

4.3 Juglans regia (Juglandaceae) - noce

The common walnut has been reported in the ethnobotanical literature for its use as an aromatic for cheese and to protect it from parasites (Guarrera et al., 2005a) in Italy, and as a medicine for rheumatism, fever, fungal infection, hemorrhoid, cough and eczema in Turkey (Erdemoglu et al., 2003; Kultur, 2007). The bark is prepared in a decoction and gargled for toothache and fresh leaves are applied topically to reduce the swelling associated with varicose veins (Quave et al., 2008). The immature fruits are used for cosmetic (hair dye) applications (Pieroni et al., 2004) and the flowers are employed in ritual healing ceremonies for mal d’arco (rainbow illness) (Quave and Pieroni, 2005).

An ethanolic extract of the immature fruits reduced biofilm adherence and formation. Limited growth inhibition was noted at higher doses 128–512 μg/ml (Figure 2c). Similar activity in staphylococcal growth inhibition by aqueous extracts of walnut leaves has also been reported (Pereira et al., 2007). The walnut tree is rich in phenolic compounds, including naphtoquinones and flavonoids that are likely responsible for the anti-bacterial activity of extracts derived from this plant (Pereira et al., 2007; Stampar et al., 2006). The use of walnut leaves for mild skin inflammations has been approved by the German Commission E (Blumenthal et al., 2000). This is the first report of anti-biofilm activity for MRSA by walnut extract.

4.4 Leopoldia comosa (Hyacinthaceae) - cipuldjin

In southern Italy, the tassel grape hyacinth is used in folk remedies for toothache (bulb is grated and applied topically) (Guarrera et al., 2005b) and for food (Casoria et al., 1999; Pieroni et al., 2002b). Extracts from the bulbs have demonstrated potent in vitro anti-oxidant activity (Pieroni et al., 2002a). Both the aqueous and ethanolic extracts of the bulb demonstrated potent anti-biofilm activity for this strain of MRSA. The IC50 for preventing biofilm formation and adherence was 16 and 8 μg/ml, respectively. The IC90 for biofilm adherence was 128 μg/ml (Figure 2d). This is the first report of anti-biofilm activity for this plant.

4.5 Marrubium vulgare (Lamiaceae) - marugg

White horehound is one of the most frequently quoted medicinal species applied in south Italian traditional medicine. A local rhyme is associated with this plant: “U’ marrugxh, ogni mal’ destrugg ” (the white horehound destroys every disease). It is used in the treatment of furuncle, boil, athlete’s foot, skin infection, dermatitis, foot and mouth disease (in animals), gastritis, and malaria. It is thought to be particularly good at cleansing the liver and is used as a hepatoprotectant and general panacea (Pieroni et al., 2002c; Pieroni and Quave, 2005). Aqueous decoctions of the aerial parts are either used to rinse the skin or are drunk, depending upon the illness being treated. An ethanolic extract of the roots was effective in reducing adherence of established biofilms at very low concentrations (IC50 = 8 μg/ml and IC90=128 μg/ml). This extract was not, however, very effective at preventing the formation of biofilms on plastic surfaces. The maximum percent inhibition for biofilm formation was 31% at a concentration of 128 μg/ml (Figure 2e).

Previous studies have demonstrated that white horehound extracts exhibit antispasmodic (Schlemper et al., 1996), antioxidant (Berrougui et al., 2006; Matkowski and Piotrowska, 2006; Weel et al., 1999), hypotensive (El Bardai et al., 2001), insecticidal (Pavela, 2004), and analgesic (Meyre-Silva et al., 2005) properties. Marrubium species are rich in diterpenes, caffeic acid derivatives, sterols, and flavonoids (Khanavi et al., 2005; Lazari et al., 1999). One of the principle medicinal components of horehound extract is marubiin, a furanic labdane diterpene (Blumenthal et al., 2000). It has been found to exhibit non-selective anti-inflammatory properties in mouse models, which may support its use as a folk remedy for dermatitis (Stulzer et al., 2006). The German Commission E approved the use of this herb for the treatment of loss of appetite and dyspepsia (Blumenthal et al., 2000). The effect of white horehound extract on biofilm formation and adherence in MRSA has not been previously reported.

5. Conclusion

Plant extracts demonstrated limited bacteriostatic activity, and in fact, many actually promoted planktonic growth. More significantly, extracts from five plants exhibited dose-dependent inhibition of biofilm formation and adherence. We identified a significant correlation in anti-biofilm activity with medicinal plants used for SSTI when compared with plants with no apparent ethnomedical use. These results validate the efficacy of the topical application of Arundo donax, Ballota nigra, Juglans regia, Leopoldia comosa, and Marrubium vulgare in Italian folk remedies for SSTI.

While these results are based on the analysis of crude, unfractionated extracts, they also represent the first of many steps towards the development of new anti-biofilm drugs. Inhibition of biofilms provides a method of controlling the effects of pathogenic bacteria without strong selection for drug resistance. The emergence of infections due to highly resistant MRSA capable of forming and many times involving biofilms presents a significant dilemma to medicine today. The healthcare community is ill-prepared to effectively treat such infections as the old classes of antibiotics currently in use rapidly lose their efficacy. Effective treatments for the disruption of established biofilms could save many lives and decrease healthcare costs related to the treatment and potential replacement of infected implanted prosthetic devices. Prophylactic agents for biofilms embedded in surgically implanted devices and catheters could significantly diminish morbidity and subsequently reduce healthcare costs associated with nosocomial infection.

We recommend further investigation of natural products from the following plants for anti-biofilm activity in MRSA: Lonicera alpigena, Castanea sativa, Juglans regia, Ballota nigra, Rosmarinus officinalis, Leopoldia comosa, Malva sylvestris, Cyclamen hederifolium, Rosa canina, and Rubus ulmifolius.

Acknowledgments

The project described was funded supported by Grant Number 1F31AT004288-01A1 from the National Center for Complementary and Alternative Medicine. The content is solely the responsibility of the authors and does not necessarily reflect the official views of the National Center for Complementary and Alternative Medicine or the National Institutes of Health. Additional support for this project was provided by MBRS RISE - NIH/NIGMS R25GM061347, Botany in Action, Anne Chatham Fellowship in Medicinal Botany, USDA CSREES 20053842215940 and NIH/NCAAM 1T32AT01060-01.

We thank Dr. Carmine Colacino and Dr. Andrea Pieroni for assisting in the taxonomic identification of plants. We also thank the Caputo family for logistical support during field research. Thanks to Dr. Roberto Perez and Prof. Steve Davis for providing bacterial isolates and training in biofilm assays. Special thanks to volunteers who assisted in laboratory work: Jana Rose, Marco Caputo and Susan Mendez. Lastly, we extend our gratitude to all of the study participants who graciously shared a wealth of knowledge regarding the traditional medical practices of their communities.

Footnotes

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