Citrus bergamia belongs to the Citreae tribe in the Aurantioideae subfamily of the Rutaceae plant family and is defined as a hybrid between a sour orange (C. aurantium L.) and lemon (C. lime L. Burm. f.) or a mutation of the latter. Trees are cultivated almost exclusively along the coast of the Calabria region, Southern Italy [1,2].

Bergamot fruit is principally used for its essential oils that are extracted from the peel of the fruit by rasping and cold-pressing procedure. Bergamot essential oil is one of the main basic constituents for the manufacture of perfumes and it is widely used in cosmetics such as in aromatherapy, mainly for reducing stress and anxiety [3]. Antiinflammatory, anti-proliferative activities [4,5] and analgesic effects [6,7] were also demonstrated.

Popular tradition has always ascribed antimicrobial properties to bergamot oil, and these features were firstly studied in the 19^th century by Francesco Calabrò, a physician coming from Calabria region [8].

At present several studied report antimicrobial effects against bacteria, yeasts, filamentous fungi, of different type of derivative from bergamot essence as well as varied bergamot oil extraction [9,10]. Focà et al. [8] has also evaluated the antiviral activity of the essential oil against Herpes Simplex type 1, clearly showing its inhibitory capacity on viral replication during single-cycle growth curves. The aim of this study was to evaluate the “in vitro” antimicrobial activity of natural essence and distilled extract of bergamot on drug resistance pathogens isolated from clinical specimens. The activities were tested using a micro well dilution assay. To verify the killing effects of the above substances on microorganisms we used alcidine orange staining procedures and conoohocal studies.

Antimicrobial activities of natural essence and distilled extract of bergamot, a kindly gift by Prof. G. Sindona from the Department of Chemistry and Chemical Technology, University of Calabria, Cosenza (Italy), were tested on a range of clinical pathogens isolated from patients admitted to University Hospital of Catanzaro, Italy, by using different methods such as a micro well dilution assay and co focal microscopy. Pathogens included Gram-positive (S. epidermidis, S. pyogenes, S. aureus, C. striatum, E. faecalis), Gram-negative bacteria (S. maltophilia, A. baumannii, K. pneumoniae, P. aeruginosa, E. coli, E. aerogenes) and yeasts (C. albicans, C. glabrata, C. lypolytica, C. tropicalis, C. lusitaniae). Strains were conventionally identified by biochemical testing (Vitek-2, bioMérieux, France). Antibiotic sensitivity was evaluated by Vitek System (bioMérieux, France).

Gas chromatography analysis

The chemical composition of tested substances (essential oil and distillate) was analyzed with Hewlett Packard (Agilent) gas chromatograph (model HP 5890A) - mass spectrometer (model HP 5972A) equipped with a HP-35MS column. Helium was used as carrier gas and ionization was obtained by electron impact. Temperature of the column was maintained at 60°C for 5 minutes, and then raised to 280°C in 10°C/minutes increments. Nineteen compounds, which were available reference standards, were identified and quantified. Other compounds were identified by comparison of mass spectra of each peak with those of authentic samples in the NIST (National Institute of Standard and Technology) library.3

Micro well dilution assay

Aliquots of 1 ml of each substance were placed in sterile glass test tubes and were emulsified with Tween 20, an inert, non ionic tensioactive agent with no inherent bactericidal and fungicidal activity (900 μl of substance plus 100 μl of Tween 20). Mixture was then vortexes and the first dilution of the emulsion was performed in a liquid medium (Nutrient Broth, BioMérieux), adding 100 ml of emulsion in 2 ml of liquid medium (dilution 1:20). 100 μl of the first dilution were dispensed into the first well of a sterile micro titration plate and twofold serial dilutions were then performed.

Microorganism’s identification was performed by biochemical testing (Vitek-2, bioMèrieux, France). The microorganism under investigation were prepared in test tubes containing sterile physiological solution, diluting colonies grown on solid medium to obtain a bacterial suspension of 0.5 McFarland (about 10⁸ colony-forming units [CFU]/ ml). Each microbial suspension was then diluted 1:100 in liquid medium (100 μl bacterial suspension in 9.9 ml Nutrient Broth, BioMérieux), and then 50 μl were added in all plate micro wells.

Each plate was incubated for 24 hours at 37°C. After incubation, to determine antimicrobial effect of tested substances, aliquots of 1 μl from each dilution, were inoculated in plates (Blood agar and Sabouraud agar) and incubated for 24 hours at 37°C, before colony counting.

In all experiments we included the substance alone to exclude possible contamination (negative control) and bacterial suspension, without substance, as positive control of microbial growth.

Confocal microscopy

To assess the bactericidal and fungicidal activity of bergamot natural essence and bergamot distilled extract, 10 μl, containing bacterial or fungal inoculums plus substance to be tested at different dilutions, was mixed with 10 μl of acridine orange, a nucleic acid selective met achromatic stain. During acridine orange staining procedures, the fluorescent dye discriminates live from dead cells giving out a green and a red fluorescence respectively. Images were acquired under a 63x objective using a confocal microscope (Leica TC-SP2 Confocal System, Leica Microsystems Srl, and Milan, Italy).

Gas chromatography analysis

The chromatograms of essential oil and distillate are reported in Figures 1 and 2, respectively. Figure 1 and Table 1 show that the major component of the essential oil was lynalyl acetate (TR=12:03 min, 42.22%). Probably this result can be attributed to the period of harvest of the fruits. Among cyclic monoterpenes mainly representative were limonene (tR=7.48 min, 24.32%), linalool (tR=9.47 min, 10.86%) and β-pinene (tR=5.99 min, 6.56%). Figure 2 and Table 1 show that in the distillate the main component was limonene (tR=7:48 min, 30.20%), followed by linalool (tR=9.47 min, 21.82%), linalyl acetate (tR=12:03 min, 16.21%) and ɣ-terpinene (tR=8:37 min, 11.95%).

Figure 1: Chromatogram of echsrsomenattoigarla pohi l( moofd bele HrPg 5a8m90oAt). - Cmahses mspieccatrlo mceotmer p(moosdietli oHnP 5o9f7 2eAs) seequniptpiaeld owiitlh o af H bPe-3r5gMaSm coolut mann. alyzed with Hewlett Packard (Agilent) gas chromatograph (model HP 5890A) - mass spectrometer (model HP 5972A) equipped with a HP-35MS column.

Figure 2: Chromatogram of dcishrtoilmleadto geraxpthr a(mcot doefl HbPe 5rg89a0mA) o- mt. aCssh sepemctircomale tceor (mmopdoels HitPio 5n97 o2Af )d eiqsutiipllpeedd weitxht ar aHcPt- 3o5fM bSe crogluammn. ot analyzed with Hewlett Packard (Agilent) gas chromatograph (model HP 5890A) - mass spectrometer (model HP 5972A) equipped with a HP-35MS column.

Table 1: Composition of essential oils and distilled extract of Bergamot.

Microwell dilution assay

The activity of different dilutions of natural essence and distilled extract of bergamot has been tested on Gram-positive and Gram-negative bacteria. In particular S. epidermidis (three strains), S. aureus (two strains), S. pyogenes (one strain), C. striatum (one strains), E. faecalis (one strain), S. maltophilia (one strain), A. baumannii (one strain), K. pneumoniae (three different strains), P. aeruginosa (one strain), E. coli (one strain), E. aerogenes (one strain) All bacteria, their isolation site as well as antibiotic resistance profile are listed in Tables 2-4.

Table 2: Isolation site and antibiotic resistance profiles of bacterial clinical isolates.

Table 3: Antimicrobial activity of essential oil of bergamot.

Table 4: Antimicrobial activity of distilled extract of bergamot.

Moreover substance activities have been tested on different Candida species such as C. albicans (three strains isolated from blood, bronchoalveolar lavage and sputum respectively), C. lypolytica (two strains both isolated from sputum), C. tropicalis isolated from blood, C. glabrata, isolated from vaginal swab and resistant to Fluconazole and Voriconazole, and C. lusitaniae isolated from bronchoalveolar lavage, also resistant to Fluconazole.

Tables 2 and 3 summarized antimicrobial activity of essential oil and distilled extract respectively. Essential oil was mostly active on a multi drug resistant Acinetobacter baumannii (up to 1:320 dilutions), moreover a slight effect on Candida spp growth was detected. Data revealed that the distilled extract of bergamot possessed greatest antimicrobial efficacy when compared with essential oil. In particular the effects were evident on S. pyogenes strain (up to 1:80 dilutions) as well as on S. epidermidis clinical isolate (up to 1:80 dilution). Concerning antifungal activity, bergamot distilled extract was effective against all yeast strains studied, in particular when it was tested on blood isolated Candida albicans strain (up to 1.2560 dilution). Finally the activity on a multi drug resistant Acinetobacter baumannii was extremely successful (up to 1:2560 dilutions). Although data differed among microorganisms, a reduction of total number of colony forming units was always observed (data not shown).

Confocal microscopy

Concerning the bactericidal/fungicidal activity, visualized by confocal microscopy during acridine orange staining procedures, images of E. coli strain, treated with bergamot distilled extract (Figure 3) and C. albicans strain, treated with bergamot distilled extract (Figure 4) are reported. Pictures show the dose dependent bactericidal/fungicidal effects on both microorganisms.

Figure 3: Confocal microscopy during acridine orange staining procedure of E. coli clinical isolate treated with bergamot distilled extract. Bactericidal activity of distilled extract of bergamot vs. E. coli (1:40 dilution) (Panel A); No Bactericidal activity (1:640 dilution) (Panel B); Positive control of E. coli growth (Panel C).

Figure 4: Confocal microscopy during acridine o(Parnael nC).ge staining procedure of C. albicans clinical isolate treated bergamot distilled extract. Fungicidal activity of distilled extract of bergamot vs. C. albicans (1:40 dilution) (Panel A); No fungicidal activity (1:640 dilution) (Panel B); Positive control of C. albicans growth (Panel C).

Our data show a more powerful activity of bergamot distilled extract when compared with bergamot essential oil, moreover bergamot distilled extract were found to be more active against Gram-negative bacteria and yeast tested.

Chemical composition of substances tested can affect the mode of action and antimicrobial activity, so the most abundant compounds, found in both mixtures, are limonene, a cyclic monoterpenes, linalool and lynalyl acetate, oxygenated acyclic monoterpenes, which together account for about 77% and 70% of their composition respectively. The major variation between the two mixtures includes difference in concentration/combination of the three components mentioned before and this condition could affect the different antimicrobial activities of the substances. On the other hand the antimicrobial activity of the monoterpenes has been previously investigated and it is related to the relative lipophilicity and water solubility, then significantly influenced by their physicochemical characteristics, is and by the composition of bacterial membranes [11]. Moreover, interaction between the different constituents can occur, causing antagonistic, additive and synergistic antimicrobial effects [12-15]. Indeed, essential oils show an antimicrobial activity greater than their main components suggesting a possible interaction between all of its constituents [16,17].

The hydrophilic permeability barrier, due to lipopolysaccharide molecole, in the outer membrane of Gram-negative bacteria, confers protection against the effects of highly hydrophobic drugs [18]. Even so, we found in vitro activity of bergamot distilled extract on drug resistant clinical isolates such as Acinetobacter baumanni, Stenotrophomonas maltophilia, Escherichia coli and Klebsiella pneumoniae. Interestingly, such bacteria cause nosocomial infection and represent a worldwide public health problem.

Notably the effectiveness on Acinetobacter baumanni (A. baumannii), an emerging pathogen responsible for community- and hospital-acquired infections that are difficult to control and treat.

It is thought that infections are acquired after exposure to A. baumannii, which is able to survive on contaminated hospital equipment’s or by contact with healthcare personnel exposed to this microorganism through contact with a colonized patient. Indeed this species can be isolated from human skin or feces as well as from hospital bed rail also many days after discharge of an infected patient from the hospital. Therefore, alternative antimicrobial substances, such as distilled extract of bergamot, could be of great importance to prevent A. baumannii contaminations on hospital devices and healthcare personnel hand skin.

Concerning antimicrobial activity against Gram-positive bacteria, it is well known that the presence of polysaccharides, at the microorganism’s capsule surface can confer a certain resistance to the microbicidal action of the substances. This could be the case of both Gram-positive and Gram-negative bacteria, like Pseudomonas aeruginosa. Indeed a S. epidermidis strain, isolated on a catheter tip and a Corynebacterium striatum strain, isolated in peritoneal fluid were the only Gram-positive microorganism inhibited.

Finally, regarding Candida spp strains, which possess the abundance of sterols on their exterior envelope, leading their chemical and physical affinity for different bergamot constituents, we confirm previously observations and suggest bergamot potential role in topical treatment of Candida infections [8].

Moreover, considering bactericidal/fungicidal activity, established through observation of dead cells by acridine orange staining, our data give a further scientific support to frequent opinion in the usefulness of treating skin infections with bergamot oils and its derivatives [19,20]. Therefore, this underlines the need for additional studies to better understand bergamot inhibition mechanisms.

We thank Prof. G. Sindona, Department of Chemistry and Chemical Technology, University of Calabria, Cosenza, Italy for the kind gift of natural essence and distilled extract of bergamot.