A M Schito

G Piatti

M Stauder

A Bisio

E Giacomelli

G Romussi

C Pruzzo

Effects of demethylfruticuline A and fruticuline A from Salvia corrugata Vahl. on biofilm production in vitro by multiresistant strains of Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis

Keywords:

Five strains of S. aureus (three meticillin-resistant and two meticillinsusceptible), five strains of S. epidermidis (four meticillin-resistant and one meticillinsusceptible) and eight vancomycin-resistant E. faecalis, all recently isolated from clinical specimens and capable of slime production, were studied. fA decrease by at least two-fold the hydrophobic properties of the S. aureus cell membrane but did not affect S. epidermidis or E. faecalis. Biofilm formation on polystyrene plates was quantified spectrophotometrically by established methodologies. Inhibition of biofilm formation was also confirmed by the Congo red agar plate assay. dfA and fA were more effective against S. aureus strains (>70% effect at subinhibitory concentrations) than against S. epidermidis in inhibiting slime synthesis. Against E. faecalis, dfA at subinhibitory concentration induced an inhibition of biofilm production of ca. 60%; fA was less active and more strain-dependent. Moreover, the two compounds were shown to posses chelating activity on divalent and trivalent metal cations.

Interactions of fA and dfA with bacteria could be very complex, possibly being species-specific, and could depend not only on inhibition of exopolysaccharide synthesis but also on their chelating activity and on changes in the microorganism's surface, including cell hydrophobicity.

A c c e p t e d M a n u s c r i p t

Introduction

Bacterial biofilms are complex and highly structured communities of sessile microorganisms embedded in a self-produced extracellular matrix of exopolysaccharide (EPS) and irreversibly attached on various surfaces [START_REF] Donlan | Biofilms: microbial life on surfaces[END_REF][START_REF] Moons | Bacterial interactions in biofilms[END_REF]. The presence of these microbial communities is often associated with various chronic diseases (including cystic fibrosis, periodontitis, chronic prostatitis, otitis media, endocarditis and recurrent urinary tract infections) [START_REF] Parsek | Bacterial biofilms: an emerging link to disease pathogenesis[END_REF] and is also responsible for the alarming spread of slime-associated nosocomial infections, mainly due to the increasing use of implanted medical devices of polymeric origin [START_REF] Otto | Staphylococcus epidermidis-the 'accidental' pathogen[END_REF]. Biofilmassociated bacteria often tolerate conditions that would eliminate their planktonic counterparts and also show great tolerance toward antibiotics and host immune defences [START_REF] Donlan | Biofilms on central venous catheters: is eradication possible?[END_REF][START_REF] Høiby | Antibiotic resistance of bacterial biofilms[END_REF]. Eradication of these communities is rarely achieved, with dire consequences for patients. Gram-positive bacteria, particularly Gram-positive cocci such as Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis, are extremely important pathogens in the hospital environment and are able to colonise a great variety of medical devices of polymeric origin [START_REF] Speziale | Prevention and treatment of Staphylococcus biofilms[END_REF][START_REF] Mccann | Staphylococcus epidermidis devicerelated infections: pathogenesis and clinical management[END_REF][START_REF] Mohamed | Biofilm formation by enterococci[END_REF].

In the European setting, the findings of the European surveillance of ICU-acquired infections (HELICS-ICU) indicated that coagulase-negative staphylococci (CoNS) were isolated in 17.5-40.7% of Intensive Care Unit-acquired bloodstream infections and that S. aureus were isolated in 7.1-19% [START_REF] Suetens | European surveillance of ICU-acquired infections (HELICS-ICU): methods and main results[END_REF]. In addition, the prevalence of S. aureus isolates resistant to meticillin and, by definition, to all -lactam drugs [meticillin-resistant S. aureus (MRSA)] was shown to range from 32.4% to 53.3% of the cases [START_REF] Suetens | European surveillance of ICU-acquired infections (HELICS-ICU): methods and main results[END_REF]. With regard to CoNS, the incidence of meticillin resistance is even A c c e p t e d M a n u s c r i p t 5 more widespread. In fact, despite marked geographic variations, in some areas of Europe >60% of CoNS are meticillin-resistant and multiresistant [START_REF] Stefani | Epidemiology of methicillin-resistant staphylococci in Europe[END_REF][START_REF]Evolution in the antibiotic susceptibility and resistance [in Italian][END_REF].

Enterococcus faecalis was also investigated in this study since it is the most common enterococcal species responsible for 80-90% of human enterococcal infections [START_REF] Jones | Emerging resistance among bacterial pathogens in the intensive care unit-a European and North American Surveillance study (2000-2002)[END_REF].

The prevalence of biofilm production observed in this pathogen is higher than that reported for Enterococcus faecium and biofilm formation is considered to represent an important factor in the pathogenesis of enterococcal infection [START_REF] Mohamed | Biofilm formation by enterococci[END_REF]. The occurrence of vancomycin-resistant enterococci, their most alarming trait, has progressively increased in the USA and in some parts of Europe over the past 5 years and it is very difficult to control [START_REF] Deshpande | Antimicrobial resistance and molecular epidemiology of vancomycin-resistant enterococci from North America and Europe: a report from the SENTRY antimicrobial surveillance program[END_REF].

This situation can be expected to further complicate treatment and potentially lead to increased morbidity and mortality. The need for new classes of antibacterial compounds with activity against multiresistant pathogens has therefore become urgent. Several reports are available describing the action of naturally occurring molecules as antimicrobial agents [START_REF] Mahady | Medicinal plants for the prevention and treatment of bacterial infections[END_REF][START_REF] Itokawa | Plant-derived natural product research aimed at new drug discovery[END_REF][START_REF] Potterat | Drug discovery and development with plant-derived compounds[END_REF]. Such plant preparations are often used as antibacterial agents in traditional medicine [START_REF] Guay | Cranberry and urinary tract infections[END_REF][START_REF] Zaidi | Bactericidal activity of medicinal plants, employed for the treatment of gastrointestinal ailments, against Helicobacter pylori[END_REF]. Terpenoids, glycosteroids, flavonoids and polyphenols are small molecules (molecular weight < 500 Da) naturally produced by plants that can inhibit many bacterial species, particularly Gram-positive organisms. These compounds are receiving sustained attention regarding their potential use since there has been strong evidence that they possess, in addition to antimicrobial activity, anti-inflammatory and antitumour properties [START_REF] Liu | Oleanolic acid and ursolic acid: research perspectives[END_REF][START_REF] Yogeeswari | Betulinic acid and its derivatives: a review on their biological properties[END_REF].

A c c e p t e d M a n u s c r i p t 6 The genus Salvia includes more than 900 species and is now being intensively studied in view of the antioxidant, antibacterial and antifungal properties that many exudation products, mainly containing ursolic and oleanolic acids, have been shown

to posses [START_REF] Kamatou | Antibacterial and antimycobacterial activities of South African Salvia species and isolated compounds from S. chamelaeagnea[END_REF][START_REF] Weckesser | Screening of plant extracts for antimicrobial activity against bacteria and yeasts with dermatological relevance[END_REF][START_REF] Kuzmaa | Antimicrobial activity of diterpenoids from hairy roots of Salvia sclarea L.: salvipisone as a potential anti-biofilm agent active against antibiotic resistant staphylococci[END_REF][START_REF] Gonzalez | A first study of antibacterial activity of diterpenes isolated from some Salvia species (Lamiaceae)[END_REF].

We recently focused our studies on an exudation product obtained from the fresh aerial part of Salvia corrugata Vahl., an American species of subgenus Calosphace, section Corrugatae, that contained several active compounds, some of which (such as ursolic and oleanolic acid) are already well characterised in their antimicrobial activity [START_REF] Weckesser | Screening of plant extracts for antimicrobial activity against bacteria and yeasts with dermatological relevance[END_REF][START_REF] Fontanay | Ursolic, oleanolic and betulinic acids: antibacterial spectra and selectivity indexes[END_REF][START_REF] Horiuchi | Antimicrobial activity of oleanolic acid from Salvia officinalis and related compounds on vancomycin-resistant enterococci (VRE)[END_REF]. We have described the strong antibacterial activity of two highly oxidised diterpene quinones, fruticuline A (fA) and demethylfruticuline A (dfA), in S. corrugata exudate against several multiresistant Gram-positive human pathogens [START_REF] Bisio | Antimicrobial activity of the ornamental species Salvia corrugata, a potential new crop for extractive purposes[END_REF]. Moreover, the biological effects dfA on mammalian cells lines were also recently investigated [START_REF] Giannoni | The administration of demethyl fruticulin A from Salvia corrugata to mammalian cells lines induces 'anoikis', a special form of apoptosis[END_REF].

The aim of the present study was to investigate further the antibacterial properties of dfA and fA by assessing whether the two molecules are able to interfere with the ability of multidrug-resistant (MDR) clinical isolates of S. aureus, S. epidermidis and E. faecalis to produce biofilms.

A c c e p t e d M a n u s c r i p t 7

Materials and methods

Microorganisms

Five strains of S. aureus (three MRSA and two meticillin-susceptible), five strains of S. epidermidis (four meticillin-resistant and one meticillin-susceptible) and eight strains of vancomycin-resistant E. faecalis, all recently isolated from clinical specimens and identified according to standard procedures [START_REF] Murray | Manual of clinical microbiology[END_REF], shown to be capable of slime production were studied.

Compounds

dfA and fA were extracted and isolated from leaf surface constituents as previously described [START_REF] Bisio | Antimicrobial activity of the ornamental species Salvia corrugata, a potential new crop for extractive purposes[END_REF]. Briefly, fresh aerial parts of S. corrugata Vahl. were immersed in CH 2 Cl 2 for 20 s. After filtration and removal of the extraction solvent, the exudate (25 g, 0.96% w/w of fresh plant) was chromatographed in portions of 1.0 g on a Sephadex TM LH-20 column using CHCl 3 /MeOH (7:3) as an eluent. The eluate fractions were combined by thin layer chromatography to obtain four main fraction groups (I-IV): group I, which was rich in waxy inactive compounds; group II, which was shown to be a crystalline mixture of ursolic acid and oleanolic acid; group III, which was extracted with hot hexane and gave a residue that crystallised from EtOH, yielded crude fA (purified by recrystallisation from CHCl 3 /EtOH); and group IV, which was crystallised from EtOH and yielded crude dfA. The amounts of the compounds obtained were 4.2 g for the mixture of ursolic and oleanolic acids, 0.76 g for fA and 2.8 g for dfA.

A c c e p t e d M a n u s c r i p t 8 Sterile stock solutions of dfA and fA in dimethyl sulphoxide (DMSO) were prepared and, following dilution in appropriate media, were employed in the studies detailed below.

Susceptibility tests

Minimal inhibitory concentrations (MICs) of the two quinones for all the strains studied were determined following the microdilution procedure detailed by the Clinical and Laboratory Standards Institute (CLSI) [START_REF]Performance standards for antimicrobial susceptibility testing; sixteenth informational supplement[END_REF] using cation-adjusted Mueller-Hinton broth (Biolife, Milan, Italy) as the test medium. Briefly, overnight cultures of bacteria were diluted to yield a final concentration of ca. 5  10 5 cells/mL. Samples were then added to equivalent volumes of various concentrations of dfA and fA distributed on a microplate and prepared from serial two-fold dilutions ranging from 0.015 mg/L to 256 mg/L. Following incubation for 24 h at 37 C, the lowest concentration of compound that prevented visible growth was recorded as the MIC. All MICs were determined in duplicate.

Hydrophobicity test

Bacterial surface hydrophobicity was evaluated according to Rosenberg [START_REF] Rosenberg | Bacterial adherence to hydrocarbons: a useful technique for studying cell surface hydrophobicity[END_REF]. Three selected representative strains (one each of S. aureus, S. epidermidis and E. faecalis) were grown overnight in appropriate media. Following incubation, cells were harvested by centrifugation (3500 rpm, 20 min), suspended in phosphate buffer (PB) and exposed to 0.5 MIC of dfA or fA for 1 h at 37 C; control suspensions were left untreated. Treated and control suspensions were then washed twice by A c c e p t e d M a n u s c r i p t 9 centrifugation at 3500 rpm for 20 min and the bacteria were re-suspended in phosphate-urea-magnesium sulphate (PUM) buffer [START_REF] Rosenberg | Bacterial adherence to hydrocarbons: a useful technique for studying cell surface hydrophobicity[END_REF]. Aliquots (1.5 mL) of the bacterial suspensions were added to 1 mL of n-octane. After allowing mixing for 120 s and settling for 10 min for the separation step, absorbance of the aqueous phase was measured at 400 nm. Hydrophobicity changes in bacterial cells were expressed as percentage of absorbance variation of the PB phase of dfA-or fA-treated versus control suspensions.

Detection of slime production by the Congo red agar (CRA) plate assay

Several strains of S. aureus and S. epidermidis were screened for qualitative slime production by the CRA plate assay as described by Freeman et al. [START_REF] Freeman | New method for detecting slime production by coagulase-negative staphylococci[END_REF]. All chemicals were supplied by Biolife. Congo red stain was prepared as a concentrated aqueous solution, autoclaved separately and added to the media when the agar had cooled to 55 C. Plates were inoculated and incubated aerobically for 24 h at 37 C. Biofilmpositive strains produced black coloured colonies, whilst biofilm-negative strains were coloured pink. The total test time was 24 h. The CRA test was also used with two representative strains of S. aureus and two of S. epidermidis to evaluate directly the effect of dfA and fA on slime production. In this latter case, dfA or fA corresponding to 0.5 MIC relative to the selected strains were added together with the Congo red to the media when the agar had cooled to 55 C. Control plates containing the Congo red stain but without dfA or fA were also prepared. Plates where then inoculated with the selected isolate and were incubated aerobically for 24 h at 37 C.

A c c e p t e d M a n u s c r i p t 10 2.6. Detection of biofilm production using the microtitre plate method Slime production was detected using the microtitre plate method, and the presence and extent of biofilm structures were quantified spectrophotometrically using a method based on that reported by Cramton et al. [START_REF] Cramton | In vitro methods to study biofilm formation[END_REF]. To produce biofilms, 25 L of stationary-phase bacterial cultures were added aseptically to the wells of a 96-well polystyrene tissue culture plate (Corning, Milan, Italy) containing 175 L of tryptic soy broth medium with 0.25% glucose and were incubated at 37 C for 24 h. To evaluate the effect of dfA and fA on biofilm synthesis, the same procedure was followed except that each compound was added to the growth medium at selected concentrations. After 24 h of exposure, media were discarded and each well was washed three times with phosphate-buffer saline to remove non-adherent cells.

Plates were air-dried in an inverted position. Adherent microorganisms were stained with crystal violet. Excess stain was rinsed off with running tap water and the plates were air-dried. Adherent bacterial films were quantified spectrophotometrically by determining the optical density at 570 nm (OD 570 ). Strains producing a biofilm with an OD 570 ≤ 0.2 were considered as negative for biofilm production and were excluded from the study. Each isolate was tested in triplicate. The total test time was 48 h. The

Chelating activity test

To demonstrate the ability of the two quinones to chelate metal ions, the similarity of the 11-oxogroup and 12-hydroxygroup of these compounds to the 4-oxogroup and 3hydroxygroup of the flavonols, which are able to chelate cations, was used. Assays for cation-chelating activity were thus performed following the methods described by Mabry et al. [START_REF] Mabry | The systematic identification of flavonoids[END_REF] and Von Steinegger and Hänsel [START_REF] Steinegger | Lehrbuch der Pharmakognosie und Phytopharmazie[END_REF]. Moreover, since Mg acetate is used to provide chelates for the quantitative ultraviolet (UV) determination of hydroxyanthracene derivatives of cathartic drugs, which contain 9-oxo-and 1hydroxy-groups [START_REF]European Pharmacopoeia[END_REF], UV spectra of the two quinones were recorded following addition of this reagent and bathochromic shifts of the UV absorption peaks were also observed. The stock solutions of fA and dfA were at a concentration of 0.1 mg/mL.

Statistical analysis

Student's t-test was used to evaluate any significant differences between the OD 570 values obtained without the two compounds (controls) and those observed in the presence of different concentrations of the two quinones.

Results

Minimal inhibitory concentrations

The results of susceptibility testing of dfA and fA for the strains studied are given in Table 1. dfA and fA displayed a very narrow distribution of MIC values, ranging from 32 mg/L to 64 mg/L and from 16 mg/L to 32 mg/L when assayed against S. aureus A c c e p t e d M a n u s c r i p t 12 and S. epidermidis, respectively. MIC values for fA and dfA were more uniform (32 mg/L each) against, respectively, S. aureus and S. epidermidis. This suggests a relatively more potent action of dfA against S. epidermidis and of fA against S. aureus. Interestingly, there was no correlation between MIC values and resistance to meticillin in all strains tested. Against E. faecalis, the two quinones displayed a very homogeneous potency, with MIC values not exceeding 64 mg/L.

Effect of dfA and fA on cell hydrophobicity

Considering that surface hydrophobicity plays a key role in the adhesion ability of biofilm-producing bacteria, the ability of fA and dfA to promote changes in the cell surface properties of the pathogens was assessed by evaluating the affinity of these bacteria following exposure to the two quinones towards n-octane. The two compounds were tested at concentrations corresponding to 0.5 MIC. fA was able to decrease strongly the hydrophobic properties of the S. aureus cell membrane, increasing more than two-fold the percentage of bacteria distributed in the aqueous phase following treatment with the compound (Table 2). In contrast, dfA did not display any effect on the cell hydrophobicity of the same pathogen (Table 2). Moreover, no changes in cell hydrophobicity following fA and dfA exposure were observed in S. epidermidis and E. faecalis (data not shown).

A c c e p t e d M a n u s c r i p t 13

Inhibition of slime synthesis determined by the Congo red agar plate assay in Staphylococcus spp.

Since inhibition of biofilm synthesis might be the result of a negative effect of the compounds on bacterial growth or on slime production or both, it was first verified that the activity of the two quinones on the growth of both bacterial species was indeed negligible at the selected sub-MIC values (data not shown).

To investigate further the role of fA and dfA in preventing biofilm synthesis in S. aureus and S. epidermidis strains, the ability of the two compounds to inhibit EPS production in the two pathogens was evaluated first by a qualitative method, the CRA plate assay. dfA and fA were used at concentrations corresponding to 0.5 MIC calculated for the slime-producing strains being testing and were incorporated into CRA plates to verify whether the developing colonies would turn from black to pink coloured. The experiments were conducted on two selected isolates of S. aureus and S. epidermidis and, since the results obtained were identical, only the patterns obtained with S. aureus are shown (Fig. 1). Both quinones were clearly able to induce production of pink-coloured colonies instead of the classical black colonies.

The effect of the two compounds was evident especially where bacterial colonies were more isolated.

Inhibition of slime synthesis determined by the microtitre plate method

To assess in a quantitative manner the antibiofilm activity of the two compounds, further experiments were conducted using the microtitre plate method. The two quinones dfA and fA displayed clear activity in preventing staphylococcal and and3). In contrast, S. epidermidis did not display the same sensitivity to fA and dfA when tested at the same sub-MIC dilutions (Figs 2 and3).

In addition, a more variable inhibitory effect of dfA with respect to fA on S. aureus strains was noted at 0.25 and 0.5 MIC concentrations (Fig. 2). This behaviour was strongly strain-dependent, irrespectively of the resistance traits possessed by the organisms, and it was not directly related to the drug concentration used. This finding confirmed that dfA displays a more powerful action against S. aureus than fA. A similar variability in the activity of dfA was also observed against S. epidermidis stains at 0.5 MIC values (Fig. 2).

The antibiofilm activity of the two quinones on E. faecalis was less prominent and was more strain-dependent than that observed against staphylococci. For dfA, the percentages of inhibition of biofilm synthesis ranged from 10.66% to 85.19% at 0.25 MIC and from 20.26% to 89.49% at 0.5 MIC for the eight strains tested. fA was active at these sub-MIC values only on five of the eight strains tested, providing an inhibition ranging from 9.35% to 64.97% at 0.25 MIC and from 17.31% to 72.54% at 0.5 MIC values. These results indicate that dfA is more potent than fA in homogenously inhibiting biofilm production in E. faecalis.

A c c e p t e d M a n u s c r i p t 15

Chelating activity test

To evaluate further the possible mechanism of the inhibitory effect of fA and dfA on biofilm formation in staphylococci, the ability of the two quinones to chelate metal ions known to be required for the synthesis and stabilisation of biofilm structures was investigated [START_REF] Otto | Staphylococcal biofilms[END_REF].

The clear bathochromic shifts observed with both reagents demonstrated that these compounds are able to produce a chelate with Al 3+ , which is stable not only in a neutral environment but also in the presence of acid. Even Mg chelation was stable at physiological range, but it was unstable following addition of HCl (results shown below

Discussion

Here we studied the biofilm-inhibiting properties of two highly oxidised diterpene quinones, fA and dfA, extracted from the surface exudate obtained from the fresh aerial part of S. corrugata Vahl., an American species of the subgenus Calosphace, that is easily grown in Mediterranean coastal areas.

These readily available compounds were shown to reduce, in different ways and with distinct intrinsic potency, the multistep process of biofilm synthesis of several MDR strains of staphylococci and enterococci.

Against staphylococci, dfA and fA displayed MIC values with a very narrow distribution, ranging from 32 mg/L to 64 mg/L and from 16 mg/L to 32 mg/L, respectively. There was no correlation between MIC values and resistance to meticillin, indicating that the two compounds are acting with a different and still uncharacterised mechanism compared with traditional antibiotic drugs. The same feature is even more true for E. faecalis, with a single MIC of 64 mg/L. The ability of the two diterpene quinones to interfere with the crucial and initial docking phase of biofilm formation, when hydrophobic interactions between the bacterial cell wall and the surface to be colonised play a vital role, was first analysed. fA at 0.5 MIC values was able to modify greatly the properties of the S. aureus surface, decreasing by at least two-fold its hydrophobic nature compared with the untreated control (Table 2). No changes in cell hydrophobicity were observed for S. Using one qualitative (CRA for staphylococci) and one quantitative method (microtitre plate for all pathogens), it was demonstrated that dfA and fA possess an inhibitory activity on several biofilm-producing and MDR Gram-positive strains tested. The effect of the two compounds was evident both against S. aureus and S. epidermidis when evaluated by the CRA qualitative method. This may suggest that the two quinones could interfere with the synthesis of polysaccharides that form the glycocalyx, disrupting their formation and thus reducing the amount of slime that accumulates [START_REF] Teather | Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen[END_REF][START_REF] Allison | The role of exopolysaccharides in adhesion of freshwater bacteria[END_REF]. Moreover, when using the quantitative method it became apparent that the inhibition produced by fA against S. aureus is not only slightly more substantial but is also more uniform compared with the activity of dfA on the same pathogen (see Figs 3 and2, respectively). This behaviour could be due to the significant reduction in the hydrophobic surface properties that fA was shown to induce specifically in S. aureus. The reduction both in cell wall hydrophobicity and in EPS production could thus contribute to the overall decrease in biofilm synthesis observed with fA in S. aureus.

The differences observed between the action of fA and dfA against S. aureus and S. The effects of fA and dfA in preventing biofilm synthesis in E. faecalis were evaluated only by the quantitative method. Against this species, the two quinones displayed a less prominent ability to inhibit biofilm production compared with that in staphylococci. For fA, a more heterogeneous effect within the isolates tested was also apparent. This latter feature may be attributed to chemical and physical heterogeneity of the specific biofilms utilised by the strains studied. Moreover, it appears that cell wall hydrophobicity is not affected in E. faecalis by the two quinones, suggesting that other mechanisms excluding modification of surface hydrophobicity may be the target of action of fA and dfA.

An alternative and attractive possibility that could explain the mechanism of the inhibitory effect of the two quinones on biofilm synthesis, particularly for dfA on S. aureus and possibly for both quinones on S. epidermidis and E. faecalis, could be attributed to the chelating effect possessed by the two compounds. It was possible to evaluate the chelating activity of the two quinones by considering that the chemical structures of fA and dfA belong to the same chemical class as horminone, an abietane diterpene quinone that can penetrate bacterial cells as a Mg 2+ chelate [START_REF] Nicolas | Theoretical study of the structure and antimicrobial activity of horminone[END_REF].

We have supposed the same activity for the two quinones and utilised the similarity of the 11-oxogroup and 12-hydroxygroup of these compounds to the 4-oxogroup and 3-hydroxygroup of the flavonols, which are able to chelate cations, to demonstrate the ability of fA and dfA to chelate metal ions. As with the flavonoids, chelation with Al 3+ is detected by bathochromic shifts of the UV absorption peaks and is used for the systematic identification of these molecules [START_REF] Mabry | The systematic identification of flavonoids[END_REF]. We thus compared the UV spectra of fA and dfA in methanol with the spectra obtained after addition of AlCl 3 and A c c e p t e d M a n u s c r i p t 19 AlCl 3 /HCl [START_REF] Mabry | The systematic identification of flavonoids[END_REF]. The results obtained demonstrated that fA and dfA are able to chelate biologically important bivalent and trivalent cations that are stable in a neutral environment.

These results indicate that interactions of fA and dfA with Gram-positive bacteria could be very complex, possibly being species-specific, and could depend not only on EPS synthesis inhibition but also on their chelating activity and on changes in the microorganism's surface, including cell hydrophobicity.

*

  Corresponding author. Tel.: +39 010 353 7753; fax: +39 010 353 7651.E-mail address: amschito@unige.it (A.M. Schito).

A c c e p t e d M a n u s c r i p t 3 ABSTRACT

 3 In this study, demethylfruticuline A (dfA) and fruticuline A (fA), two quinones representing the mayor diterpenoid components of the exudate produced by the aerial parts of Salvia corrugata, were assessed for their ability to modify surface characteristics, such as hydrophobicity, and to inhibit synthesis of biofilm in vitro by multiresistant Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis.

  results were derived from three separate experiments and OD 570 values were expressed as mean ± standard deviation. The OD 570 value obtained for each strain without any added compound was used as the control. The percentages of biofilm formed in the presence of different concentrations of dfA and fA were calculated employing the ratio between the values of OD 570 with and without the compound, adopting the following formula: [(OD 570 with drug/OD 570 without drug)  100].

A

  c c e p t e d M a n u s c r i p t 14 enterococcal biofilm synthesis. Staphylococcus aureus strains were more susceptible to the inhibitory effect of both compounds compared with S. epidermidis (see Figs 2

A c c e p t e d M a n u s c r i p t 17 aureus

 17 when treated with dfA or when of the two quinones were tested against S. epidermidis or E. faecalis.

  epidermidis are probably due to their diverse chemical structure, depending in particular on the absence of the methyl group in dfA. It should be underlined that within these two diterpene quinones, even a minor change in the structural configuration of the molecule (a demethylation in the case of dfA) can greatly modify the functional activity of the compound on bacteria cell wall hydrophobicity.

Fig. 1 .

 1 Fig. 1. Bacterial colonies of a selected strain of Staphylococcus aureus (F) grown on Congo red agar plates, showing decreasing levels of slime production in the presence of sub-MICs of demethylfruticuline A (dfA) or fruticuline A (fA): (A) black

Fig. 2 .

 2 Fig. 2. Activity of demethylfruticuline A (dfA) against five Staphylococcus aureus and five Staphylococcus epidermidis strains (% inhibition).

Fig. 3 .

 3 Fig. 3. Activity of fruticuline A (fA) against five Staphylococcus aureus and fiveStaphylococcus epidermidis strains (% inhibition).

  s c r i p t

  ). fA: UV/Vis  max (CH 3 OH) 210, 251, 277, 321, 390sh nm; (CH 3 OH + AlCl 3 ) 210, 259,

	287, 318, 424 nm; (CH 3 OH + AlCl 3 + HCl) 210, 259, 287, 318, 424 nm; (CH 3 OH + Mg
	acetate) 226, 254, 282, 331, 400 nm; (CH 3 OH + Mg acetate + HCl) 231, 254, 276,
	319, 400 nm.
	dfA: UV/Vis  max (CH 3 OH) 208, 254, 278, 319, 399sh nm; (CH 3 OH + AlCl 3 ) 207, 258,
	288, 317, 426 nm; (CH 3 OH + AlCl 3 + HCl) 207, 255, 290, 318, 420 nm; (CH 3 OH + Mg
	acetate) 229, 254, 279, 329, 401 nm; (CH 3 OH + Mg acetate + HCl) 233, 254, 283,
	323, 399 nm.

Table 1

 1 Minimal inhibitory concentrations (MICs) of demethylfruticuline A (dfA) and fruticuline A (fA) for Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus

	faecalis
	Microorganism (n) Strain MIC (mg/L)
			dfA	fA
	S. aureus (5)	A a	32	32
		B a	64	32
		D	64	32
		F	32	32
		G a	32	32
	S. epidermidis (5) 1 a	32	32
		2 a	32	32
		3 a	32	16
		4	32	32
		7 a	32	32
	E. faecalis (8) b	14	64	64

Table 2

 2 Effect of demethylfruticuline A (dfA) and fruticuline A (fA) at 0.5 the minimal inhibitory concentration on Staphylococcus aureus surface hydrophobicity % aqueous phase S.D.

	Control 36.4	7.9
	dfA	32.2	12.0
	fA	89.6	11.9
	S.D., standard deviation.

Funding

This study was supported by a grant of EU INTERREG ALCOTRA project 'AROMA no. 68'.

Competing interests

None declared.

Ethical approval

Not required.

A c c e p t e d M a n u s c r i p t a Indicates meticillin resistance.

b All enterococci were vancomycin-resistant and multiresistant.

Edited Table 1 A c c e p t e d M a n u s c r i p t Edited Figure 1


G N Ngameni

J R Folefoc

M W Ouahouo

V Kuete

S Alibert-Franco

K O Eyong

B Ngameni

G N Folefoc

J R Nguemeving

J G Tangmouo

G W Fotso

J Komguem

B M W Ouahouo

J.-M Bolla

J Chevalier

B T Ngadjui

A E Nkengfack

J.-M Pagès

J.- M Bolla

Transporteurs Membranaires

Antibacterial activity of some natural products against bacteria expressing a multidrug-resistant phenotype

Keywords:

Antibacterial activity of some natural products against

bacteria expressing a multidrug-resistant phenotype

A c c e p t e d M a n u s c r i p t

Introduction

Multidrug resistance is a worrying cause of treatment failure in bacterial infections [START_REF] Blot | Measuring the impact of multidrug resistance in nosocomial infection[END_REF][START_REF] Falagas | Pandrug-resistant Gram-negative bacteria: the dawn of the post-antibiotic era?[END_REF][START_REF] Chopra | Treatment of health-care-associated infections caused by Gram-negative bacteria: a consensus statement[END_REF]. Various widely used antibiotics favour the selection of bacteria resistant to multiple antibacterial agents [START_REF] Nikaido | Multidrug resistance in bacteria[END_REF]. The expression of mutated genes, coding for resistance to a single drug (e.g. mutation in a target) or a specific resistance mechanism (e.g. enzymatic barrier), in association with the modulation of multidrug efflux pump or porin expression involved in transport, are the most common processes described in multidrug-resistant (MDR) bacteria [START_REF] Nikaido | Multidrug resistance in bacteria[END_REF][START_REF] Alekshun | Molecular mechanisms of antibacterial multidrug resistance[END_REF][START_REF] Bolla | Membrane permeability and regulation of drug 'influx and efflux' in enterobacterial pathogens[END_REF]. A numbers of chemicals, such as phenylalanine arginine -naphthylamide (PAN) and 1-(1-naphthylmethyl)piperazine, quinoline derivatives, etc. [START_REF] Pagès | Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria[END_REF], as well as natural products [START_REF] Stavri | Bacterial efflux pump inhibitors from natural sources[END_REF] have been found to inhibit bacterial efflux pumps. In addition, several natural compounds and integral components belonging to the usual pharmaceutical library have been tested to combat resistant bacteria [START_REF] Fischbach | Antibiotics for emerging pathogens[END_REF][START_REF] Saleem | Antimicrobial natural products: an update on future antibiotic drug candidates[END_REF]. The aim of the present work was to determine the antibacterial activities and potencies of several molecules, including terpenoids, alkaloids and phenolic compounds, against different bacterial strains expressing a MDR phenotype. The role of efflux pumps was investigated using pump-deleted strains and the efflux pump inhibitor (EPI) PAN. Furthermore, this study aimed to determine the pharmacophoric group present in the active compounds.

Material and methods

Chemicals for antimicrobial assays

Chloramphenicol

Bacterial strains and culture media

MDR isolates and reference strains of Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa and Klebsiella pneumoniae are summarised in Table 1.

Isolates were conserved at 4 C and were grown on Mueller-Hinton agar for 24 h before minimal inhibitory concentration (MIC) testing. Mueller-Hinton broth (MHB) was used for the susceptibility tests [START_REF] Kuete | Antitumor, antioxidant and antimicrobial activities of Bersama engleriana (Melianthaceae)[END_REF].

Determination of bacterial susceptibility

The respective MICs of the various compounds against the bacteria were determined using a rapid XTT colorimetric assay [START_REF] Viveiros | Inducement and reversal of tetracycline resistance in Escherichia coli K-12 and expression of proton gradient-dependent multidrug efflux pump genes[END_REF]. Briefly, samples were first dissolved in dimethyl sulphoxide (DMSO)/MHB. The solution obtained was then added to MHB A c c e p t e d M a n u s c r i p t and was sequentially diluted two-fold in a 96-well microplate. Then, 100 L of fresh inoculum (1.5  10 6 colony-forming units/mL) prepared in MHB was added. The plates were covered with a sterile plate sealer, homogenised and incubated at 37 C for 18 h. The concentration of DMSO used did not affect microbial growth. Wells containing MHB, 100 L of inoculum and DMSO was used as a negative control (systematically added). Chloramphenicol was used as a reference antibiotic. MICs were measured after 18 h incubation at 37 C, followed by addition of 40 L of 0.2 mg/mL INT and incubation at 37 C for 30 min. Viable bacteria reduced the yellow dye (INT) to pink. The MIC was defined as the lowest concentration of compound that prevented this change and exhibited complete inhibition of microbial growth. Samples were tested alone or in the presence of PAN at a final concentration of 20 mg/L. All assays were carried out in triplicate and were repeated three times.

Results

Studied compounds

The different natural compounds investigated in the present study are shown in Fig.

The terpenoids included four diterpenoids (hardwickiic acid and crotozambefurans

A-C) and three pentacyclic triterpenoids (3-friedelanone, betulinic acid and oleanolic acid). Two alkaloids (norcassaide and norerythrosuaveolide) were tested. Phenolic compounds, including four coumarins, five xanthones, nine quinones and seven flavonoids, were also investigated. The compounds were tested for their ability to prevent the growth of MDR and reference strains of Gram-negative bacteria, alone and in the presence of the EPI PAN. The results are summarised in Table 2.

A c c e p t e d M a n u s c r i p t

Activity of the terpenoids

Both the diterpenoids and the triterpenoids exhibited very weak activities. Tested alone, none of them was active against any of the tested bacterial strains (Table 2).

The presence of EPI conferred only a little activity to some of the compounds against AcrAB-or TolC-deleted strains.

Activity of the alkaloids

Norcassaide and norerythrosuaveolide showed a moderate and limited activity against the tested bacteria. An MIC value of 64 mg/L was obtained with norcassaide against E. coli AG100A, E. aerogenes EA294 and EA298 and the reference P. aeruginosa PA01 strain (Table 2). A corresponding value for norerythrosuaveolide of 32 mg/L was noted against E. coli AG100A. A significant decrease in MIC values was observed in the presence of the EPI PAN.

Activity of phenolics (coumarins, xanthones, quinines and flavonoids)

Most of the coumarins presented a reduced and limited activity when tested alone.

The best activity (MIC = 32 mg/L) was obtained with MAB3 against E. coli AG100A.

Most of the xanthones were poorly active. Laurentixanthone B showed significant activity, with its antibacterial effects being observed on all investigated bacteria including MDR strains. The quinone plumbagin, the two chalcones (flavonoids) 4hydroxylonchocarpin and bartericin C, and MAB3 (coumarin) were found to be active against bacteria including MDR strains. MIC values <10 mg/L were obtained for plumbagin against E. coli AG100A and AG100A Tet . The activity of this compound was better than that of chloramphenicol (MIC = 32 mg/L) against AG100A Tet . 4-

A c c e p t e d M a n u s c r i p t

Hydroxylonchocarpin and bartericin C showed interesting antimicrobial properties, but the two compounds had an MIC > 10 mg/L against the bacteria studied in this work.

Role of efflux pumps in the susceptibility of tested bacteria: identification of PAN-sensitive efflux in addition to the AcrAB-TolC pump

To assess the involvement of efflux in the activity of the tested compounds, the compounds were combined with the well known EPI PAN. The results are summarised in parentheses in Table 2. When tested alone none of the terpenoids were active against K. pneumoniae or P. aeruginosa (Table 2), whereas a reduced and limited activity was noted with crotozambefuran B and C against E. aerogenes EA294 and EA298. Despite this moderate activity, it is important to note that an antibacterial effect of the tested terpenoids was detected against the efflux-deleted strain E. coli AG100A, except for crotozambefuran A. However, the enhancing effect of the EPI was limited during combination with terpenoids. PAN significantly increased the activity of most of the tested phenolic compounds. For the coumarins, MICs < 10 mg/L were recorded with MAB3 against four E. coli strains and E. aerogenes EA298. Interestingly, PAN increased the activity of laurentixanthone B, and MIC values < 10 mg/L were noted for two E. coli strains (Table 2). Amongst the quinones, plumbagin (naphthoquinone), which was active against all studied bacteria when tested alone, showed a significant increase in activity in the presence of the EPI. MICs < 10 mg/L were obtained with studied bacteria including MDR and reference strains. Furthermore, this activity was quite similar to or better than that of chloramphenicol in several cases. It should also be noted that one of the most active flavonoids (4-hydroxylonchocarpin) also exhibited MIC values < 10 mg/L against A c c e p t e d M a n u s c r i p t several MDR bacteria (Table 2). It is important to note the effect of the EPI on the susceptibility of E. coli and E. aerogenes strains deleted of AcrAB and TolC components (Table 2). This suggests the involvement of PAN-sensitive efflux in addition to the AcrAB-TolC efflux pump. When tested in the presence of the EPI, compounds were active in ca. 46% (94/204), 54% (37/68), 64% (65/102) and 75% (127/170) of the cases against E. aerogenes, P. aeruginosa, K. pneumoniae and E. coli, respectively, whilst they were only active in 29%, 41%, 48% and 46% of the cases when tested alone.

Structure-activity relationship study

When exploring the structure-activity relationship, it can be noted that terpenoids (both diterpenoids and triterpenoids) are poor antimicrobial compounds. A careful examination of the activities of the two tested alkaloids (norcassaide and norerythrosuaveolide with a terpenoid-like ring) suggests that the presence of nitrogen in the structure may enhance the antimicrobial activity of terpenoids.

Regarding the activity of coumarins, it could be noted that 7-methoxycoumarin is generally more active than 7-hydroxycoumarin against MDR Gram-negative bacteria.

1,5-Dihydroxyxanthone and 1,7-dihydroxyxanthone exhibited similar activities, indicating that the location of the hydroxyl group in the structure does not significantly modulate their activities. It is also important to note that a simple naphthoquinone (plumbagin) exhibited better activity compared with 4-hydroxylonchocarpin (flavonoid), laurentixanthone B (xanthone) and MAB3 (coumarin). From a molecular structure point of view, the natural products exhibiting the best antibacterial activities have the same pharmacophore (Fig. 2). As plumbagin is the minimal scaffold required to have significant antibacterial activity in the absence of an EPI, it could be A c c e p t e d M a n u s c r i p t suggested that this pharmacophore is involved in the antibacterial activity. Other functional groups and their position (Fig. 2), in addition top those of the pharmacophoric moiety, may affect the antibacterial properties and modulate the susceptibility of the molecule to bacterial resistance mechanisms.

In the presence of the EPI, MIC values < 10 mg/L were obtained in 2 (12.5%), 5 (31.3%), 9 (56.3%) and 16 (100%) of the 16 tested bacteria, respectively, for laurentixanthone B, MAB3, 4-hydroxylonchocarpin and plumbagin (Table 2).

Discussion

Tripartite drug efflux pumps, mainly those clinically reported as AcrAB-TolC in Enterobacteriaceae or as MexAB-OprM in P. aeruginosa, play a key role in multidrug resistance of pathogenic Gram-negative bacteria [START_REF] Nikaido | Multidrug resistance in bacteria[END_REF][START_REF] Bolla | Membrane permeability and regulation of drug 'influx and efflux' in enterobacterial pathogens[END_REF]. PAN has been reported as a potent inhibitor of the resistance-nodulation-cell division (RND) efflux systems and is particularly active on AcrAB-TolC and MexAB-OprM [START_REF] Pagès | Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria[END_REF][START_REF] Lomovskaya | Practical applications and feasibility of efflux pump inhibitors in the clinic-a vision for applied use[END_REF]. To determine the involvement of efflux pumps, the concentration of PAN used in this study (20 mg/L) had no intrinsic effect on the bacteria as previously determined [START_REF] Lorenzi | Geraniol restores antibiotic activities against multidrug-resistant isolates from Gram-negative species[END_REF], i.e. no significant variation in the MIC of chloramphenicol was observed for AG100A (AcrAB-deleted derivative of AG100). In contrast, under these conditions a significant increase in the antibacterial activity of several compounds was noted, showing that a number of natural products are substrate of efflux pumps acting in resistant strains of E. coli, E. aerogenes, K. pneumoniae and P. aeruginosa. However, this was not the general case with terpenoids, indicating that either this class of secondary metabolites is not highly active or that RND efflux pumps are not the main mechanism of resistance involved against such compounds in these MDR bacteria. Terpenoids could also A c c e p t e d M a n u s c r i p t have some difficulties in passing through the bacterial membrane barrier. The enhancing effect of PAN observed against the studied MDR bacteria with various compounds such as laurentixanthone B, plumbagin, 4-hydroxylonchocarpin, bartericin C and MAB3 suggests that PAN-sensitive efflux could be a major means of resistance. Regarding the involvement of MDR bacteria in treatment failures and the re-emergence of infectious diseases [START_REF] Blot | Measuring the impact of multidrug resistance in nosocomial infection[END_REF][START_REF] Falagas | Pandrug-resistant Gram-negative bacteria: the dawn of the post-antibiotic era?[END_REF][START_REF] Nicolle | Infection control programmes to contain antimicrobial resistance[END_REF], the activity of some compounds (in the presence of PAN) could be considered very promising. Pseudomonas aeruginosa is an important nosocomial pathogen highly resistant to clinically used antibiotics, causing a wide spectrum of infections and leading to substantial morbidity and mortality [START_REF] Cardoso | Surveillance of antimicrobial susceptibility of Pseudomonas aeruginosa clinical isolates from a central hospital in Portugal[END_REF]. MDR Enterobacteriaceae, including K. pneumoniae, E. aerogenes and E. coli, have also been classified as antimicrobial-resistant organisms of concern in healthcare facilities [START_REF] Nicolle | Infection control programmes to contain antimicrobial resistance[END_REF]. Therefore, laurentixanthone B, plumbagin and 4hydroxylonchocarpin could be attractive candidates for the development of a new antimicrobial family against MDR strains in combination with EPIs. The antimicrobial activities of bartericin C and crotozambefurans A-C are being reported and the results of the present study provide a clear explanation of the low bacterial susceptibility previously observed for other compounds [START_REF] Mbaveng | Antimicrobial activity of the extracts and compounds from Vismia guineensis (Guttiferae)[END_REF][START_REF] Kuete | Diospyrone, crassiflorone and plumbagin: three antimycobacterial and antigonorrhoeal naphthoquinones from two Diospyros species[END_REF]. The studied compounds included terpenoids, phenolics and alkaloids. Within the phenolic compounds, some of them, such as laurentixanthone B, MAB3 and 4hydroxylonchocarpin, exhibited significant activity and contained the same pharmacophore as observed in plumbagin.

By analysing the role of efflux mechanisms in the antimicrobial activity of the various compounds, it can be observed that compounds were more active against the tested bacteria in the presence of an EPI. These data clearly draw attention to the Minimal inhibitory concentrations (MICs) of the studied components against the tested bacterial species PAN, phenylalanine arginine -naphthylamide; N/T, not tested.

A c c e p t e d M a n u s c r i p t

i i i (16) i i i i i i i i i i i i i Crotozambefuran A i i i i i i i i i i i i i i i i Crotozambefuran B i i i (256) i i i i i i (128) i (128) i i i i i i Crotozambefuran C i (128) i i (128) i i i i i i (128) i (128) i i i i i i 3-Friedelanone i i i (128) i i i i i i i i i i i i i Betulinic acid i i i (256) i i i i i i i i i i i i i Oleanolic acid i (128) i i (128) i i i i i i i i i i i i i Coumarins MAB3 i (32) 128 (8) 32 (4) i (8) i (8) 256 (64) i i (32) i (16) i (8)
a The MIC of PAN was 64 mg/L for AG100A and >256 mg/L for other E. coli, E. aerogenes, K. pneumoniae and P. aerogenes strains.

i, sample not active up to 256 mg/L.

A

  c c e p t e d M a n u s c r i p t ABSTRACT The present study assessed the antimicrobial activities of various natural products belonging to the terpenoids, alkaloids and phenolics against a collection of Gramnegative multidrug-resistant (MDR) bacteria. The results demonstrated that most of the compounds were extruded by bacterial efflux pumps. In the presence of the efflux pump inhibitor phenylalanine arginine -naphthylamide (PAN), the activities of laurentixanthone B (xanthone), plumbagin (naphthoquinone), 4-hydroxylonchocarpin (flavonoid) and MAB3 (coumarin) increased significantly against all studied MDR bacteria. Laurentixanthone B, 4-hydroxylonchocarpin and MAB3 contained the same pharmacophoric moiety as plumbagin. This study indicates that the AcrAB-TolC (Enterobacteriaceae) and MexAB-OprM (Pseudomonas aeruginosa) efflux pumps are involved in resistance of Gram-negative bacteria to most of the natural products.

  (Sigma-Aldrich, St-Quentin-Fallavier, France) was used as a reference antibiotic molecule. p-Iodonitrotetrazolium chloride (INT) and PAN were A c c e p t e d M a n u s c r i p t used as a microbial growth indicator and EPI, respectively. Natural products (Fig. 1) were obtained from the chemical bank of the Laboratory of Organic Chemistry, University of Yaoundé I, Cameroon. Isolation and identification of the following compounds have been reported previously [10-13]: crotozambefurans A, B and C; bartericin C; MAB3; mammeisin; norcassaide; norerythrosuaveolide; 1,8-dihydroxy-6methoxy-3-methylanthraquinone; 3-geranyloxy-6-methyl-1,8-dihydroxyanthraquinone; vismiaquinone and vismiaquinone C; bivismiaquinone; laurentixanthone B; kaempferol; lapachol; 2-acetylfuro-1,4-naphathoquinone; newbouldiaquinone and newbouldiaquinone A; hardwickiic acid; 3-friedelanone; betulinic acid; oleanolic acid; 7-methoxycoumarin; 7-hydroxycoumarin; 1,5-dihydroxyxanthone; smeathxanthone A and smeathxanthone B; gancaonin Q; stipulin; angusticornin B; 1,7dihydroxyxanthone; 4-hydroxylonchocarpin; and quercetin.

A

  c c e p t e d M a n u s c r i p t involvement of efflux pumps AcrAB-TolC or MexAB-OprM[START_REF] Pagès | Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria[END_REF][START_REF] Saleem | Antimicrobial natural products: an update on future antibiotic drug candidates[END_REF][START_REF] Lorenzi | Geraniol restores antibiotic activities against multidrug-resistant isolates from Gram-negative species[END_REF][START_REF] Papadopoulos | Role of the MexAB-OprM efflux pump of Pseudomonas aeruginosa in tolerance to tea tree[END_REF] in the resistance of Gram-negative bacteria to natural compounds. This study highlights the key role of efflux mechanisms against several phenolic and alkaloid compounds and, consequently, indicates that inhibition of such efflux pumps could significantly improve their antimicrobial potencies.

  s c r i p t alternifolia) oil and its monoterpene components terpinen-4-ol, 1,8-cineole and terpineol. Appl Environ Microbiol 2008;74:1932-5.

Fig. 1 .

 1 Fig. 1. Chemical structures of the natural compounds tested.

Fig. 2 .

 2 Fig. 2. Representation of pharmacophoric groups detected in the tested compounds.

A c c e p t e d M a n u s c r i p t 1 2

 12 Table

  SampleMIC (mg/L) (with 20 mg/L PAN a )

Table 1

 1 Bacterial strains and features

	Bacterial strain	Relevant features	Reference
	Escherichia coli
	ATCC 8739 and	Reference strains
	ATCC 10536
	AG100	Wild-type E. coli K-12	[14]
	AG100A	AG100 acrAB::KAN R	[14]
	AG100A Tet	acrAB mutant of AG100; TET R owing to acrF	[14]
		gene markedly overexpressed
	AG102	AG100 overexpressing AcrAB pump	[15]
	Enterobacter aerogenes
	ATCC 13048	Reference strain
	EA-CM64	CHL R variant obtained from ATCC 13048	[16]
		overexpressing AcrAB pump
	EA27	Clinical MDR isolate exhibiting energy-dependent	[17]
		NOR and CHL efflux; KAN R AMP R NAL R STR R
		TET R
	EA289	KAN-sensitive derivative of EA27	[17]
	EA294	EA289 acrA::KAN R	[17]
	EA298	EA289 tolC::KAN R	[17]
	Klebsiella pneumoniae
	ATCC 11296	Reference strain
	Kp55	Clinical MDR isolate, TET R AMP R ATM R CEF R	[18]
	Kp63	Clinical MDR isolate, TET R CHL R AMP R ATM R	[18]
	Pseudomonas aeruginosa
	PA01	Reference strain
	PA124	MDR clinical isolate	[19]
	KAN, kanamycin; TET, tetracycline; CHL, chloramphenicol; NOR, norfloxacin; AMP,
	ampicillin; NAL, nalidixic acid; STR, streptomycin; ATM, aztreonam; CEF, cefalothin;

R

, resistant; MDR, multidrug-resistant.

Acknowledgments

The authors are grateful to C.A. Elkins and L. Amaral for the gift of bacterial strains.

They would also like to thank A. Davin, A. Lieutaud, E. Goemaere, A. Molitor, H.M. Poumale and P. K. Lunga for their helpful advice and fruitful discussions.

Funding

'Agence universitaire de la Francophonie' (AUF) to VK and 'Université de la Méditerranée and Service de Santé des Armées'.

Competing interests

None declared.

Ethical approval

Not required.

A c c e p t e d M a n u s c r i p t


Mark I Garvey

Mukhlesur Rahman

Simon Gibbons
email: simon.gibbons@pharmacy.ac.uk

Laura J V Piddock
email: l.j.v.piddock@bham.ac.uk

Medicinal plant extracts with efflux inhibitory activity against Gramnegative bacteria

Keywords:

It was hypothesised that extracts from plants that are used as herbal medicinal products contain inhibitors of efflux in Gram-negative bacteria. Extracts from 21 plants were screened by bioassay for synergy with ciprofloxacin against Salmonella enterica serotype Typhimurium, including mutants in which acrB and tolC had been inactivated. The most active extracts, fractions and purified compounds were further examined by minimum inhibitory concentration testing with five antibiotics for activity against Enterobacteriaceae and Pseudomonas aeruginosa. Efflux activity was determined using the fluorescent dye Hoechst 33342. Eighty four extracts from 21 plants, 12 fractions thereof and 2 purified molecules were analysed. Of these, 12 plant extracts showed synergy with ciprofloxacin, 2 of which had activity suggesting efflux inhibition. The most active extract, from Levisticum officinale, was fractionated and the two fractions displaying the greatest synergy with the five antibiotics were further analysed. From these two fractions, falcarindiol and the fatty acids oleic acid and linoleic acid were isolated. The fractions and compounds possessed antibacterial activity especially for mutants lacking a component of AcrAB-TolC.

However, no synergism was seen with the fractions or purified molecules, suggesting that a combination of compounds is required for efflux inhibition. These data indicate that medicinal plant extracts may provide suitable lead compounds for future development and possible clinical utility as inhibitors of efflux for various Gram-negative bacteria.

A c c e p t e d M a n u s c r i p t

Introduction

Over the last decade there has been a dramatic reduction in the number of pharmaceutical companies developing new antimicrobial agents [START_REF] Boucher | Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America[END_REF]. In parallel, the number of antibiotic-resistant Gram-negative bacteria has increased [START_REF] Livermore | Has the era of untreatable infections arrived?[END_REF]. With the reduction in the number of new agents and in antibiotic development, there has been a resurgence of interest in the search for compounds that will restore the activity of licensed antimicrobial agents that until recently had excellent activity against Gramnegative bacteria. In 1998 it was shown that plant-derived compounds have activity against Gram-positive bacteria, in particular Staphylococcus aureus [START_REF] Hseih | Bacteria lacking a multidrug efflux pump: a sensitive tool for drug discovery[END_REF]. Several compounds, such as reserpine, behave as if they inhibit efflux pumps and hence have become known as efflux pump inhibitors (EPIs) [START_REF] Neyfakh | Efflux-mediated multidrug resistance in Bacillus subtilis: similarities and dissimilarities with the mammalian system[END_REF]. Since that time, numerous phytochemicals have been shown to have activity against S. aureus or other Grampositive bacteria, or to act as potential EPIs with antimicrobials for Gram-positive bacteria [START_REF] Tegos | Multidrug pump inhibitors uncover remarkable activity of plant antimicrobials[END_REF][START_REF] Stermitz | Polyacylated neohesperidosides from Geranium caespitosum: bacterial multidrug resistance pump inhibitors[END_REF][START_REF] Belofsky | Phenolic metabolites of Dalea versicolor that enhance antibiotic activity against model pathogenic bacteria[END_REF][START_REF] Belofsky | Metabolites of the 'smoke tree', Dalea spinosa, potentiate antibiotic activity against multidrugresistant Staphylococcus aureus[END_REF][START_REF] Cherigo | Inhibitors of bacterial multidrug efflux pumps from the resin glycosides of Ipomoea murucoides[END_REF][START_REF] Kumar | Novel structural analogues of piperine as inhibitors of the NorA efflux pump of Staphylococcus aureus[END_REF][START_REF] Timmer | Pelargonium sidoides extract for acute respiratory tract infections[END_REF]. Unfortunately, most of the plant-derived compounds have little or no activity with antibiotics against Gram-negative bacteria and it was suggested that, as many plant pathogens are Gram-negative bacteria, plants may not produce molecules effective against these organisms [START_REF] Tegos | Multidrug pump inhibitors uncover remarkable activity of plant antimicrobials[END_REF]. None the less, there are many medicinal plants that have traditional usage in various parts of the world in the treatment of infections caused by Gram-negative bacteria [START_REF] Rüegg | 3-Farnesyl-2-hydroxybenzoic acid is a new anti-Helicobacter pylori compound from Piper multiplinervium[END_REF][START_REF] Sawer | The effect of cryptolepine on the morphology and survival of Escherichia coli, Candida albicans and Saccharomyces cerevisiae[END_REF].

Gram-negative bacteria have innate multidrug resistance to many antimicrobial compounds owing to the presence of efflux pumps. In the Enterobacteriaceae, the efflux pump most commonly associated with this innate multidrug resistance is the AcrAB-TolC efflux system. Pseudomonas aeruginosa, Acinetobacter spp. and Campylobacter jejuni [START_REF] Piddock | Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria[END_REF]. The presence of these pumps and their broad substrate profile is the cause of the innate resistance to many of the agents that have good antimicrobial activity against Grampositive bacteria. Overproduction of these efflux pumps confers clinically relevant resistance to many antimicrobial agents, including ciprofloxacin and tigecycline, in Enterobacteriaceae [START_REF] Piddock | Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria[END_REF].

We hypothesised that extracts from plants that are used as natural herbal medicines contain molecules that act as EPIs against the efflux pumps of Gram-negative bacteria. Therefore, 84 extracts from 21 medicinal plants were investigated for synergistic activity with ciprofloxacin against Salmonella enterica serotype Typhimurium. These plants were chosen as they all have a use in various systems of medicine and some are used as topical antimicrobials, for example Melissa officinalis. Extracts were screened for differential activity against strains that either expressed or overproduced the AcrAB-TolC pump but had no activity for mutants in which a component of this pump had been inactivated. In this way, it was hoped to identify extracts that contained an EPI specific for this pump. For those extracts where data suggested active components, the effect upon growth of Salmonella spp.

as well as inhibition of efflux activity was determined. Two extracts showing the greatest synergy with ciprofloxacin were fractionated and the active molecules were identified.

A c c e p t e d M a n u s c r i p t

Materials and methods

Bacterial strains, storage and growth

All bacteria used in this study are listed in Table 1. Construction of mutants derived from S. Typhimurium has been described previously [16,19,[START_REF] Eaves | Expression of acrB, acrF, acrB, marA and soxS in Salmonella enterica serovar Typhimurium: role in multiple antibiotic resistance[END_REF][START_REF] Webber | The global consequence of disruption of the AcrAB-TolC efflux pump in Salmonella enterica includes reduced expression of SPI-1 and other attributes required to infect the host[END_REF]. Salmonella Typhimurium L3 is a pre-therapy human clinical isolate and L10 is a post-therapy human clinical isolate that overexpresses acrAB, both of which have been previously described [20,[START_REF] Piddock | Ciprofloxacin resistance in clinical isolates of Salmonella typhimurium obtained from two patients[END_REF]. All bacteria were stored on Protect TM beads (Technical Service Consultants Ltd., Heywood, UK) at -80 C. Identification of each species was confirmed by Gram-stain and analytical profile index (API 20E; bioMérieux, Marcyl'Étoile, France).

Media and chemicals

Bacteria were grown on Luria-Bertani (LB) agar plates (Oxoid Ltd., Basingstoke, UK) and in LB broth (Oxoid Ltd.). All antibiotics and other compounds were obtained from Sigma (Poole, UK).

Extraction of plants

In total, 21 plants were used in this study (Table 2). All plants except Catha edulis (purchased locally in London, UK) were purchased from Proline Botanicals (Essendine, UK). Each plant (200-400 g) was extracted at 21 C sequentially with 300-600 mL of chloroform, methanol and water.

A c c e p t e d M a n u s c r i p t 6

Fractionation of extracts

Following assessment of bioactivity, the chloroform extract of Levisticum officinale (10 g) was fractionated by vacuum liquid chromatography (VLC) on silica gel 60G (Merck Ltd., Lutterworth, UK) using solvent systems of increasing polarity. Fractions were eluted with hexane containing increasing 10% or 20% increments of ethyl acetate (EA) to give fractions A-H (A, 10% EA; B, 20% EA; C, 30% EA; D, 40% EA; E, 50% EA; F, 60% EA; G, 80% EA; and H, 100% EA). Fraction I was eluted with 5% methanol in EA, fraction J with 10% methanol in EA, fraction K with 20% methanol in EA and finally fraction L with 50% methanol in EA.

Purification of active compounds

Bioassay-directed isolation indicated that the VLC fraction eluted with 30% EA was active. This was purified by preparative thin-layer chromatography (Si Gel; Merck) (mobile phase 20% EA in toluene; double development) yielding falcarindiol (1; 450 mg) and levistolide A (2; 69 mg) (Fig. 1), which were identified by comparison with literature data [START_REF] Lechner | The antistaphylococcal activity of Angelica dahurica (Bai Zhi)[END_REF][START_REF] Kaouadji | Three additional phthalide derivatives, an epoxymonomer and two dimers from Ligusticum wallichii rhizomes[END_REF].

Bioassay screening

Screening with ciprofloxacin in the absence or presence of plant extracts (Table 2), fractions of L. officinale and M. officinalis, and purified compounds from fractions of the chloroform extract of L. officinale for synergistic activity was carried out by bioassay essentially as described previously [START_REF] Lund | Rapid gentamicin bioassay using a multiple-antibiotic-resistant strain of Klebsiella pneumoniae[END_REF][START_REF] Piddock | Natural and synthetic compounds such as trimethoprim behave as inhibitors of efflux in Gram-negative bacteria[END_REF]. Six rows of 6 wells (total 36 wells) each of 8-mm diameter were cut out of each agar plate using an agar cutter (Fisher Scientific, Loughborough, UK). Then, 200 L of sterile Iso-Sensitest TM broth A c c e p t e d M a n u s c r i p t 7 (Oxoid Ltd.) containing ciprofloxacin (0.5, 1 and 2 mg/L) was added to three sets of wells. This volume and concentration gave a zone of inhibition 15 mm in diameter.

To determine test compound synergism with ciprofloxacin, 200 L of a solution containing ciprofloxacin (0.5, 1 and 2 mg/L) and the plant extract (100 mg/L) in broth was added to three separate wells. The plates were incubated overnight at 37 C and the diameters of the inhibition zones were measured. Each experiment contained three technical repeats and was repeated on three separate days. A zone of inhibition of the antibiotic plus test plant extract larger than that of the antibiotic alone was taken to suggest synergy. Positive controls were ciprofloxacin plus Lphenylalanyl-L-arginyl--naphthylamide (PAN) (100 mg/L) or carbonyl cyanide mchlorophenylhydrazone (CCCP) (100 M), and negative controls were Iso-Sensitest broth alone and plant extract alone to determine whether the plant extract had inherent antimicrobial activity.

Determination of the minimum inhibitory concentration (MIC) of antibiotics  synergising extracts, fractions and compounds

The MICs of ciprofloxacin, tetracycline, chloramphenicol, erythromycin and ethidium bromide (EtBr) in the absence or presence of plant extracts and fractions (at 100 mg/L) were determined by the agar doubling dilution method and by the microbroth dilution method [START_REF] Andrews | Determination of minimum inhibitory concentrations[END_REF][START_REF] Andrews | Determination of minimum inhibitory concentrations[END_REF]. MIC determinations were repeated at least three times in independent experiments. Antibiotics, compounds and EPIs were made up and used according to the manufacturer's instructions.

A c c e p t e d M a n u s c r i p t 8

Determination of the fractional inhibitory concentration index (FICI)

Activity of antibiotics plus the plant extract/fraction/purified compound was determined using the checkerboard technique [START_REF] Eliopoulos | Antimicrobial combinations[END_REF]. The first antibiotic (ciprofloxacin) of a combination was serially diluted along the ordinate of a 96-well microtitre tray; the second agent (e.g. plant extract) was diluted along the abscissa. An inoculum equal to a 0.5 McFarland turbidity standard was prepared from each strain in LB broth. Each well in the microtitre tray was inoculated with 100 mL of the bacterial suspension (at 5  10 5 colony-forming units/mL) and the plates were incubated at 37 C for 24 h under aerobic conditions. The resulting checkerboard contained each combination of ciprofloxacin and test agent with wells that contained the highest concentration of each agent at opposite corners. The MIC was defined as the lowest concentration of antibiotic that completely inhibited growth of the organism as detected with the naked eye [START_REF] Andrews | Determination of minimum inhibitory concentrations[END_REF]. Synergy was defined as an FICI (FICI) of 0.5, no interaction was defined as an FICI of >0.5-4 and antagonism was defined as an FICI >4 [START_REF] Odds | Synergy, antagonism, and what the chequerboard puts between them[END_REF].

Accumulation of Hoechst 33342  plant extracts by Salmonella Typhimurium

Efflux activity of five S. Typhimurium strains [L3, L10, SL1344, ATCC 15277 (LT2) and ATCC 14028s] was determined by measuring accumulation of the fluorescent dye Hoechst 33342 (bisbenzimide; 2.5 M) in the absence or presence of known efflux inhibitors (CCCP 100 M and PAN 100 mg/L) and in the absence or presence of those plant extracts (at 100 mg/L) revealed to have activity in the bioassay and MIC tests. Measurements were taken at excitation and emission wavelengths of 350 nm and 460 nm, respectively, over 30 min using a FLUOstar A c c e p t e d M a n u s c r i p t 9 OPTIMA (BMG Labtech, Aylesbury, UK) as previously described [START_REF] Coldham | A 96-well plate fluorescence assay for assessment of cellular permeability and active efflux in Salmonella enterica serovar Typhimurium and Escherichia coli[END_REF]. Differences in accumulation in the absence of the plant extracts were analysed for statistical significance using the two-tailed Student's t-test. P-values 0.05 were considered significant.

Growth kinetics  plant extracts

The growth kinetics of L3, L10, SL1344, ATCC 15277 (LT2) and ATCC 14028s in the absence or presence of plant extracts was determined by monitoring the optical density at 600 nm every 10 min at 37 C for 24 h using a FLUOstar OPTIMA (BMG Labtech). Levisticum officinale chloroform extract and M. officinalis methanol extract (at 100 mg/L) were added to the bacterial cultures at mid-logarithmic growth phase (2 h). Samples were also removed and visualised microscopically to detect any gross changes to cell morphology, i.e. filamentation. A two-tailed Student's t-test was used to compare the generation times. P-values 0.05 were considered significant.

Results

Medicinal plant extracts synergise with ciprofloxacin

Plants were extracted with chloroform, methanol and water and were screened to identify those that showed synergy with ciprofloxacin and could be putative EPIs.

Ciprofloxacin was chosen as increased MICs of this agent are often associated with overproduction of the AcrAB-TolC pump. The positive control was PAN at 100 mg/L, which was the lowest concentration to elicit an effect in this assay. Initial and Pulmonaria officinalis (chloroform and methanol) (Table 3) as well as the chloroform extracts of Angelica sinensis, Tussilago farfara (methanol) and Commiphora molmol (chloroform) (data not shown). Of the extracts investigated, those from M. officinalis and L. officinale were the most active against strains that either had wild-type levels of AcrAB-TolC or that overproduced AcrAB (Table 4).

Chloroform and methanol extracts of L. officinale and M. officinalis (both 100 mg/L) had no effect on the growth kinetics of the five Salmonella strains tested (data not shown), and by MIC determination the extract of L. officinale had synergistic activity with ciprofloxacin against most of the strains (Table 4).

Plant extracts synergise with several antibiotics

Owing to their promising activity, the MICs of the chloroform extract of L. officinale and the methanol extract of M. officinalis (i.e. those extracts that gave the largest zones of inhibition in the bioassay) were determined in combination with tetracycline, None the less, EPI-like activity was detected for both extracts combined with tetracycline, erythromycin and EtBr against wild-type Salmonella and the strain in which AcrAB was overproduced. The MIC experiments with the chloroform extract of L. officinale indicated that it had synergistic activity with ciprofloxacin and tetracycline for the salmonellae and two Klebsiella pneumoniae strains (Table 4). Of interest, the chloroform extract of L. officinale also had synergistic activity with ciprofloxacin and EtBr for Salmonella Typhimurium in which TolC was not produced and with EtBr for S. Typhimurium in which AcrAB was not produced (Table 4). The M. officinalis extract had synergistic activity with most antibiotics for the salmonellae and K. pneumoniae strains (Table 4).

The extracts of the other plants showing synergistic activity with ciprofloxacin in the bioassay did not reduce the MIC of ciprofloxacin as much as the extracts from L. officinale or M. officinalis, and when synergy was observed it was seen for fewer bacterial strains. For these reasons, no further experiments were performed with these extracts.

A c c e p t e d M a n u s c r i p t 12

Activity of fractions derived from the chloroform extract of Levisticum officinale

As the chloroform extract of L. officinale showed synergistic activity with more agents and bacteria than that from M. officinalis, it was fractionated as the first step to the identification of the active component(s). The chloroform extract of L. officinale was subjected to chromatography and 12 fractions (A-L) were obtained. MICs of ciprofloxacin and tetracycline in the absence or presence of each fraction (100 mg/L) were determined. Compared with the chloroform extract of L. officinale, few of the fractions had the same synergistic activity with ciprofloxacin. Of interest, fractions A, C, F, G and K synergised with ciprofloxacin for the Salmonella mutants in which AcrAB and TolC were not produced, and A, C, G and K also synergised with ciprofloxacin for the two K. pneumoniae strains. Fractions A, G and K also had synergistic activity with ciprofloxacin for wild-type Salmonella and the clinical isolate that overproduced AcrAB (Table 5). Two fractions (C and F) also had antimicrobial activity alone for the mutant Salmonella in which a component of AcrAB-TolC was not produced (Table 5); this was most marked for fraction C.

Activity of compounds purified from fraction C of Levisticum officinale

As fraction C showed synergistic activity in combination with ciprofloxacin and also antimicrobial activity alone especially against the strains in which acrB and tolC had been inactivated, this fraction was analysed to identify the active compounds.

Nuclear magnetic resonance (NMR) revealed that it contained predominantly the antibacterial fatty acids oleic acid and linoleic acid plus falcarindiol and levistolide A (Fig. 1). These compounds were further purified and the spectral data were in close agreement to those published [START_REF] Lechner | The antistaphylococcal activity of Angelica dahurica (Bai Zhi)[END_REF][START_REF] Kaouadji | Three additional phthalide derivatives, an epoxymonomer and two dimers from Ligusticum wallichii rhizomes[END_REF]. The absolute stereochemistry of falcarindiol A c c e p t e d M a n u s c r i p t 13 was determined using a modified Mosher's ester method as the 3(S), 8(S) diastereomer (Fig. 1).

MICs of linoleic acid and falcarindiol were >500 mg/L for the wild-type Salmonella SL1344 and ATCC 14028s. For the TolC mutants of these strains the MIC of linoleic acid was 500 mg/L, and the MIC of falcarindiol was 75 mg/L and 125 mg/L, respectively. When linoleic acid was combined with ciprofloxacin MICs of 0.008 mg/L and 0.004 mg/L were obtained for the TolC mutants, and for falcarindiol in combination with ciprofloxacin MIC values <0.001 mg/L were obtained. Synergistic activity between ciprofloxacin and the compounds for the mutants was confirmed in checkerboard assays, with FICI values of 0.14-0.27 for the combination and the TolC mutants (Table 6). The FICI for ciprofloxacin in combination with the methanol-soluble extract from M. officinalis against L3 (pre-therapy isolate) was lower than that for L10 (that overexpresses acrB). This suggests that a higher concentration of the putative EPI is required to inhibit growth of L10 owing to the greater production of AcrB by L10. No synergy was seen with this combination for the parental strains, despite it being clearly demonstrated with the extract itself. These data suggest that an EPI-like molecule(s) was lost during the separation process or was inactivated during purification.

Efflux activity of plant extracts

Accumulation of Hoechst 33342 has been used as a marker for efflux as it is fluorescent and is a substrate of several efflux pumps including AcrAB-TolC [17,[START_REF] Coldham | A 96-well plate fluorescence assay for assessment of cellular permeability and active efflux in Salmonella enterica serovar Typhimurium and Escherichia coli[END_REF]. reduced efflux) by 1.5-2-fold (Fig. 2). The efflux activity of the strain that overexpressed AcrB (L10) was also reduced by 1.5-fold.

An increase in the amount of Hoechst 33342 accumulated in the presence of an EPI

Discussion

Many conventional drugs have arisen from natural products, including plants. However, few antimicrobial agents have come from this source, with the vast majority in clinical use derived from products naturally produced by microorganisms [START_REF] Newman | Natural products as a source of new drugs over the last 25 years[END_REF]. None the less, the use of medicinal and herbal plant remedies to treat infectious diseases is common in many countries [START_REF] Ankli | Yucatec Mayan medicinal plants: evaluation based on indigenous uses[END_REF]. Owing to the continued clinical pressure for novel approaches to combat antibiotic-resistant bacterial infections, there is a need to identify new agents to treat such infections. One approach is to screen for natural products from plants. Plants have already successfully yielded compounds with activities suggesting that they inhibit efflux pumps of Gram-positive bacteria [START_REF] Hseih | Bacteria lacking a multidrug efflux pump: a sensitive tool for drug discovery[END_REF][START_REF] Tegos | Multidrug pump inhibitors uncover remarkable activity of plant antimicrobials[END_REF]. Therefore, in this study we sought to identify medicinal plants that could provide compounds for further antimicrobial drug development. In addition, as there are many clinically licensed antibacterial agents for the treatment of infections by Gram-negative bacteria, but which are effluxed by the various pumps possessed by these bacteria, we sought to screen for activity that suggested efflux inhibition. One A c c e p t e d M a n u s c r i p t 15 desirable property of a putative EPI is that it should synergise with antibiotics for bacteria with wild-type or overproduction of AcrAB-TolC but have no affect on a strain in which AcrAB or TolC are produced. As ciprofloxacin is a substrate of many bacterial efflux pumps [START_REF] Piddock | Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria[END_REF], the initial experiments used a simple bioassay to identify plant extracts that synergised with this agent [START_REF] Piddock | Natural and synthetic compounds such as trimethoprim behave as inhibitors of efflux in Gram-negative bacteria[END_REF]. This allowed many plant extracts made under different conditions to be screened with speed. In a prior study, we sought to identify compounds with EPI-like activity and to use this as an aid to predict the common structural features of EPIs and so guide the type of plant from which to make extracts for this study [START_REF] Piddock | Natural and synthetic compounds such as trimethoprim behave as inhibitors of efflux in Gram-negative bacteria[END_REF]. Of the 84 extracts from the 21 plants, 2 extracts, an ethanol extract from M. officinalis (lemon balm) and a chloroform extract of L. officinale (lovage root), had the greatest activity in terms of antibiotic potentiation with either the antibiotics ciprofloxacin or tetracycline or the dye EtBr and these data suggest that these extracts contain an inhibitor of efflux. The extract from L. officinale had the greatest EPI-like activity and so this was taken forward and fractionated as an initial step to identify the compounds with EPI-like activity. It was surprising to find that none of the 12 fractions of the chloroform extract of L. officinale had EPI-like activity, as those fractions that displayed synergy with ciprofloxacin did so in all strains irrespective of the presence or absence of a functional AcrAB-TolC system. These data suggest an interaction with other pumps or another mechanism of synergy. Most interesting was that two fractions (C and F) had some antimicrobial activity alone against all strains including those that lacked TolC. This is highly unusual as the majority of plant extracts display activity only towards Gram-positive bacteria. Fraction C, which had the greatest antimicrobial activity, was further fractionated to identify the compounds conferring the observed antimicrobial activity.

Of these, falcarindiol (1) had the greatest antimicrobial activity and a strong additive A c c e p t e d M a n u s c r i p t 16 effect to the activity of ciprofloxacin. Falcarindiol has previously been shown to possess antibacterial activity, but for mycobacteria and Gram-positive bacteria and not Gram-negative bacteria [START_REF] Lechner | The antistaphylococcal activity of Angelica dahurica (Bai Zhi)[END_REF][START_REF] Stavri | The antimycobacterial constituents of dill (Anethum graveolens)[END_REF][START_REF] Schinkovitz | Antimycobacterial polyacteylenes from Levisticum officinale[END_REF][START_REF] Chou | Antibacterial activity of components from Lomatium californicum[END_REF]. It is possible that the activity in the fraction was potentiated by the co-isolated fatty acids, and these could have membrane permeabilising activity. As these are ubiquitous in all organisms, this does not entirely explain the data or why so few plant extracts are active against Gramnegative bacteria. Rather, the data suggest that falcarindiol is a substrate of TolC and this may be why it has not been previously identified as having antimicrobial activity for Gram-negative bacteria.

Despite not successfully obtaining the compound(s) that conferred the EPI-like activity in the original plant extract, we postulate that the putative inhibitor of efflux was either lost or was inactivated during fractionation or that it acts in combination with another compound in the plant extract from which it was separated during the fractionation process. Further work to examine reconstituting fractions is required to understand whether a synergistic phenomenon is associated with selected fractions. This will also allow us to conclude whether a putative EPI is being lost in the separation process or is unstable.

Gram-negative plant pathogens such as Pseudomonas syringae can cause enormous damage to crop plants [START_REF] Arnold | Evolution of microbial virulence: the benefits of stress[END_REF], and P. syringae has been shown to possess a homologue of the tripartite resistance-nodulation-cell division (RND) efflux system MexAB-OprM [START_REF] Stoitsova | Characterization of the RNDtype multidrug efflux pump MexAB-OprM of the plant pathogen Pseudomonas syringae[END_REF]. This would suggest that plants may not produce chemicals that are active against efflux pumps of Gram-negative plant pathogens. However, the current data suggest that plants do produce molecules with activity against Gram-A c c e p t e d M a n u s c r i p t negative bacteria. Furthermore, we postulate that these molecules are substrates of TolC (and homologues thereof) and associated efflux pumps. Therefore, for plants to withstand infection by these pathogens they need to produce EPIs for the natural antimicrobial agents to be active. It is tempting to speculate that falcarindiol is produced alongside a compound(s) that inhibits efflux.

Lorenzi et al. workers [START_REF] Lorenzi | Geraniol restores antibiotic activities against multidrug-resistant isolates from Gram-negative species[END_REF] have recently shown that extracts from plants can also provide compounds with EPI-like activity or antibacterial activity per se. They also found that an extract of an essential oil from a Corsican plant, Helichrysum italicum, was able to reduce the MIC of chloramphenicol for Enterobacter aerogenes, Acinetobacter baumannii and P. aeruginosa. Two fractions of this essential oil and one compound within this oil, geraniol, synergised with chloramphenicol both for the wild-type strain of E. aerogenes and its AcrAB mutant. Another recent study by Adonizio et al. [START_REF] Adonizio | Attenuation of Pseudomonas aeruginosa virulence by medicinal plants in a Caenorhabditis elegans model system[END_REF] showed that medicinal plant extracts can attenuate the virulence of P. aeruginosa when explored in the Caenorhabditis elegans model system.

Although the extracts were from medicinal plants previously used in herbal medicine, the active compounds were not identified. The current data taken together with that of Adonizio et al. [START_REF] Adonizio | Attenuation of Pseudomonas aeruginosa virulence by medicinal plants in a Caenorhabditis elegans model system[END_REF] and Lorenzi et al. [START_REF] Lorenzi | Geraniol restores antibiotic activities against multidrug-resistant isolates from Gram-negative species[END_REF] indicate that plants as a source of new and/or novel antimicrobial compounds with activity for Gram-negative bacteria are an underexplored resource. Plant extracts such as those described by ourselves and others provide lead compounds for further exploration and development as antimicrobial agents, as agents to inhibit efflux or for combination with licensed antimicrobials. a Details of strains are given in Table 1.

b Bold text indicates synergistic combinations (i.e. MICs for the combination lower than for the antibiotic alone).

A c c e p t e d M a n u s c r i p t a Details of strains are given in Table 1.

b Bold text indicates synergy [START_REF] Odds | Synergy, antagonism, and what the chequerboard puts between them[END_REF].

A c c e p t e d M a n u s c r i p t 4 Homologues

 4 of this pump are found in other Gram-negative bacteria, including

  A c c e p t e d M a n u s c r i p t 10 experiments were performed with Salmonella Typhimurium as a model Gramnegative bacterium and because a defined isogenic set of mutants that lack or overproduce the AcrAB-TolC pump was available. Eighty-four extracts from 21 plants were screened for synergistic activity with ciprofloxacin against S.Typhimurium SL1344, ATCC 15277 (LT2), ATCC 14028s, L3 and L10. Of these, 12 extracts showed synergy with ciprofloxacin for these strains, whereas 72 showed little or no activity.The most active antibiotic-potentiating extracts were those from L. officinale (extracted with chloroform, methanol and water), M. officinalis (chloroform and methanol), Cinnamomum aromaticum (chloroform), Pimpinella anisum (chloroform)

  A c c e p t e d M a n u s c r i p t 11 chloramphenicol, erythromycin and EtBr. All extracts showed better activity in liquid media than in agar, with MIC values one to two dilutions lower from the microbroth dilution procedure than with agar (data not shown). No antimicrobial activity was detected at 100 mg/L of either extract; likewise, chloroform and methanol had no activity at the concentration used. Synergy between the extracts from M. officinalis and L. officinale with tetracycline, chloramphenicol, erythromycin or EtBr was seen for fewer strains than when the plant extracts were combined with ciprofloxacin.

  indicates inhibition of efflux [17]. To determine whether synergistic activity displayed by the extracts of L. officinale with various antibiotics was due to inhibition of efflux, accumulation of Hoechst 33342 in the presence and absence of 100 mg/L of the chloroform and methanol extracts of L. officinale was carried out for five strains of S. Typhimurium [SL1344, ATCC 15277 (LT2), ATCC 14028s, L3 and L10]. For the four antibiotic-susceptible wild-type strains of S. Typhimurium, both extracts of L. officinale increased the amount of Hoechst 33342 accumulated (i.e.

Fig. 1 .

 1 Fig. 1. Structures of falcarindiol (1) and levistolide A (2).

Fig. 2 .

 2 Fig. 2. Accumulation of Hoechst 33342 in the absence or presence of extracts of

Table 1

 1 Bacterial strains used in this study Typhimurium, Salmonella enterica serotype Typhimurium; ATCC, American Type Culture Collection; MDR, multidrug-resistant; NCTC, National Culture Type Collection (Health Protection Agency, Colindale, London, UK).

	Strain Species	Description

2

S.

Table 2

 2 List of medicinal plants used in this study

	Plant name	Common name	Family
	Anemone nemorosa	Wood anemone	Ranunculaceae
	Angelica sinensis	Female ginseng	Apiaceae
	Apium graveolens	Dill	Apiaceae
	Artemisia abrotanum	Southernwood	Asteraceae
	Asclepias tuberosa	Butterfly weed	Apocynaceae
	Boswellia serrata	Salai	Burseraceae
	Catha edulis	Khat	Celastraceae
	Centella asiatica	Gotu kola	Apiaceae
	Cinnamomum aromaticum Cassia cinnamon	Lauraceae
	Citrus aurantium	Bitter orange	Rutaceae
	Commiphora molmol	Myrrh	Burseraceae
	Daucus carota	Wild carrot	Apiaceae
	Ephedra funereal	Death Valley ephedra Ephedraceae
	Glycyrrhiza glabra	Liquorice	Leguminosae
	Levisticum officinale	Lovage	Apiaceae
	Melissa officinalis	Lemon balm	Lamiaceae
	Papaver somniferum	Opium poppy	Papaveraceae
	Pimpinella anisum	Anise	Apiaceae
	Pulmonaria officinalis	Lungwort	Boraginaceae
	Thymus vulgaris	Thyme	Lamiaceae
	Tussilago farfara	Coltsfoot	Asteraceae

Table 3

 3 Plant extracts with greatest synergy with ciprofloxacin Pulmonaria officinalis; MeOH, methanol extraction; CHCl 3 , chloroform extraction; water, water extraction; S, synergy (zone of inhibition reproducibly larger than that of antibiotic alone); NE, no effect.

	Strain a	PAN Lo	Lo	Lo	M	M	C	Pi	Pu	Pu
			MeOH	CHCl 3	Water	MeOH	CHCl 3	CHCl 3	CHCl 3	CHCl 3	MeOH
	S. Typhimurium SL1344	S	NE	NE	NE	NE	NE	NE	NE	NE	NE
	S. Typhimurium ATCC	S	S	S	S	S	NE	S	S	S	S
	15277 (LT2)
	S. Typhimurium ATCC	S	S	S	S	S	S	S	S	S	S
	14028s
	Human pre-therapy isolate	S	S	S	S	S	S	S	S	S	S
	L3
	Human post-therapy isolate	S	S	S	S	S	S	NE	S	S	S
	L10

a Details of strains are given in Table

1

.

PAN, L-phenylalanyl-L-arginyl--naphthylamide; Lo, Levisticum officinale; M, Melissa officinalis; C, Cinnamomum aromaticum; Pi, Pimpinella anisum; Pu,

Table 4

 4 Minimum inhibitory concentrations (MICs) of ciprofloxacin (CIP), tetracycline (TET), chloramphenicol (CHL), erythromycin (ERY) and ethidium bromide (EtBr) in the absence and presence of Levisticum officinale and Melissa officinalis extracts against Gram-

	negative bacteria
	Strain a	MIC (mg/L) b
		CIP	CIP +	CIP +	TET TET	TET	CHL CHL	CHL	ERY ERY	ERY	EtBr EtBr	EtBr
			Lo	M		+ Lo	+ M		+ Lo	+ M		+ Lo	+ M	+ Lo	+ M
	S. Typhimurium	0.03 0.008 0.015 2	1	1	8	8	8	512 512	512	2048 2048 2048
	L354
	S. Typhimurium	0.03 0.008 0.008 16	0.5	0.5	8	8	8	512 256	512	2048 1024 2048
	L828
	S. Typhimurium	0.008 0.004 0.008 4	0.5	0.5	1	1	1	8	8	8	16	8	16
	L829
	S. Typhimurium	0.008 0.008 0.008 4	0.5	0.5	2	8	8	64	64	64	64	32	64
	L831
	S. Typhimurium	0.008 0.002 0.008 4	0.5	0.25 8	4	8	512 512	512	512 512	512
	L3
	S. Typhimurium	0.06 0.03	0.06	8	4	4	32	16	32	512 256	512	2048 1024 2048
	L10

Table 5

 5 Minimum inhibitory concentrations (MICs) of ciprofloxacin (CIP) in the absence and presence of the six most active fractions derived from the plant extract from Levisticum officinale against a variety of Salmonella and Gram-negative bacteria

	Strain b	MIC (mg/L) b
		CIP	CIP + Lo CIP + A CIP + C C alone CIP + F F alone CIP + G CIP + J CIP + K
	S. Typhimurium L354	0.03 0.008	0.03	0.03	250	0.03	250	0.03	0.03	0.03
	S. Typhimurium L884	0.008 0.008	0.002	0.002	64	0.008	250	0.004	0.008	0.004
	S. Typhimurium L110	0.008 0.008	0.002	0.004	128	0.008	250	0.004	0.008	0.004
	S. Typhimurium L109	0.015 0.008	0.002	0.002	8	0.008	128	0.004	0.0015 0.004
	S. Typhimurium L828	0.03 0.008	0.015	0.03	250	0.03	250	0.03	0.03	0.03
	S. Typhimurium L829	0.008 0.004	<0.001 <0.001 8	0.004	128	0.004	0.015	0.004
	S. Typhimurium L830	0.008 0.008	<0.001 <0.001 128	0.004	250	0.004	0.008	0.004
	S. Typhimurium L831	0.008 0.008	<0.001 <0.001 64	0.004	250	0.004	0.008	0.004
	S. Typhimurium L3	0.008 0.002	<0.001 <0.001 250	0.008	250	0.008	0.008	0.008
	S. Typhimurium L10	0.06 0.03	0.03	0.12	250	0.12	250	0.03	0.12	0.03
	Enterobacter cloacae A1	0.12 0.06	0.12	0.12	250	0.12	250	0.12	0.12	0.12
	Serratia marcescens B14	0.06 0.06	0.06	0.06	250	0.06	250	0.06	0.06	0.06
	Pseudomonas aeruginosa G1 1	1	1	1	250	1	250	1	1	1
	Klebsiella pneumoniae H42	0.06 0.03	0.06	0.015	250	0.015	250	0.015	0.015	0.015
	K. pneumoniae H43	0.12 0.06	0.12	0.03	250	0.03	250	0.06	0.03	0.03

.

  Typhimurium, Salmonella enterica serotype Typhimurium; Lo, chloroform extract of L. officinale; CIP + letter, indicates the fraction of the chloroform extract of L. officinale.

1 Table 6

 16 Fractional inhibitory concentration index (FICI) values of ciprofloxacin (CIP) with plant extracts, fractions and purified molecules for

	Salmonella enterica serotype Typhimurium
	Strain a	FICI b
		CIP + Lo CIP + M CIP + FC CIP + FF CIP + Lin CIP + Fal
	SL1344	0.94	0.97	0.99	0.97	1.34	0.97
	SL1344 tolC::aph			0.27	0.38	0.17	0.26
	ATCC 15277 (LT2)	0.46	0.59
	ATCC 14028s	0.48	0.41	1.01	0.98	1.09	1.05
	ATCC 14028s ∆tolC			0.32	0.33	0.14	0.14
	Human pre-therapy isolate L3	0.32	0.13
	Human post-therapy isolate L10 0.31	0.22

Lo, Levisticum officinale chloroform extract; M, Melissa officinalis methanol extract; FC, fraction C from L. officinale; FF, fraction F from L. officinale; Lin, linoleic acid; Fal, falcarindiol.

Acknowledgments

Funding

This work was supported by The Leverhulme Trust (project F/00/094/AR).

a Details of strains are given in Table 1.

b Bold text indicates synergistic combinations (i.e. MICs for the combination lower than for the antibiotic alone).