Mark R Harris

Peter J Coote

Combination of caspofungin or anidulafungin with antimicrobial peptides results in potent, synergistic killing of Candida albicans and Candida glabrata in vitro

Keywords: echinocandins, antifungals, synergy, combination therapy, candidiasis Echinocandins Antifungals Synergy Combination therapy Candidiasis

Administering synergistic combinations of antifungals could be a route to overcome problems with toxicity and the development of resistance. Combination of the echinocandins, caspofungin or anidulafungin, with a range of structurally-diverse antimicrobial peptides resulted in potent, synergistic killing of Candida species in vitro. Fungicidal synergy was measured by calculating FIC values from checkerboard assays and loss of viability. Inhibitory combinations of the antifungals did not induce cytotoxicity in vitro. However, in a murine-model of systemic candidiasis, coadministration of caspofungin with one example of the cationic peptides tested, ranalexin, did not show enhanced efficacy compared to the single treatments alone. Further study using alternative peptides will identify whether this combination approach could represent a novel treatment for fungal pathogens.

Introduction

A c c e p t e d M a n u s c r i p t 3 Over the past thirty years fungi have emerged as significant causes of human disease with attendant morbidity and mortality [8,9]. A large proportion of these cases occur in immunocompromised patients because fungal pathogens are readily able to overwhelm the already weakened host defence mechanisms. Hospitalised patients with serious underlying disease are increasingly exposed to invasive surgical procedures, or chemotherapeutic treatments, such as broad-spectrum antibiotics, or immunosupressants after organ transplantation and cancer chemotherapy that readily promote mycotic disease. Infections caused by Candida albicans have a population incidence of between 1.1 and 24 cases per 100 000 individuals with an associated mortality rate of greater than 30% [10,29]. In the USA this equates to between 3 000 and 11 000 deaths annually from nosocomial candidaemia [23]. Notably, treatment with many antifungal drugs is complicated by: high toxicity and low tolerability; ineffectiveness against new or re-emerging fungi; or the development of drug-resistant strains in patients undergoing treatment [23]. Thus, there is a justifiable need for more research to develop new antifungal treatments.

One possible approach to improve the range and scope of antifungal therapy is the use of combinations of antifungals. Benefits could include: improved efficacy over the use of single drugs, consequently allowing lower, less costly, doses of each individual drug; reduced toxicity and improved tolerance; broader spectrum of efficacy; and finally, the potential to prevent resistant organisms arising as can occur with monotherapy.

A potential source of new antifungals is cationic antimicrobial peptides that are ubiquitous throughout nature and protect against invasive microbial pathogens. To date, more than 800 have been described [11]. Potential advantages of cationic peptides for future therapy include; a broad spectrum of activity, rapid killing of microbes, unaffected by classical antibiotic resistance mechanisms, neutralise endotoxin and are active in animal models [11]. Disadvantages include; sensitivity to proteolytic degradation by serum proteases in vivo; potential toxicity at high doses necessary to show efficacy in vivo, including haemolytic activity; and the high cost of synthesising and producing large quantities of these molecules [13]. Employing cationic peptides in Echinocandins inhibit the synthesis of β-D-glucan in fungal cell walls and are fungicidal against Candida and fungistatic against Aspergillus species due to loss of cell wall integrity [7]. Importantly, an intact cell wall also represents a potential barrier that cationic peptides must interact with and pass through before they can interact with the plasma membrane beneath. Supporting this, recent data from our laboratory has demonstrated that disruption of the outer cell-wall phosphomannan content has a significant influence on the degree of susceptibility of Candida albicans to a cationic peptide [12]. Thus, due to the complementary inhibitory action of the echinocandins and cationic peptides, we reasoned that the application of combinations of these molecules could result in additive or synergistic inhibition of fungal pathogens. In this study, we combined the three clinicallyapproved echinocandins with a range of structurally diverse antimicrobial peptides and observed potent, synergistic killing of Candida species in vitro.

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

Material and Methods

Yeast strains and growth media.

Yeast strains used in this work were; Candida albicans SC5314; and clinical isolates of C. albicans and C. glabrata provided by Dr. Cyril Lafong, Fife Area Laboratory, Victoria Hospital, Kirkcaldy. All strains were cultured in Malt Extract Broth (MEB), pH 7 (1% glucose, 0.6% malt extract, 0.12% yeast extract) at 30 o C with shaking. For enumerating cell survival after exposure to antifungals, yeasts were plated on YEPD agar (2% glucose, 2% agar, 1% bactopeptone, 1% yeast extract).

Numbers of cells used in the various assays described below were calculated using an optical density (OD 600 ) versus cell numbers calibration curve for each yeast strain used (data not shown).

Antifungals.

Magainin 2 [31], ranalexin [3], cyclic peptide 6752 [6], cyclic peptide GS14K4 [14], gomesin [24] and dermaseptin S3(1-16) [18] were synthesized according to their published sequences by Peptide Research Ltd, Wickham, UK, to greater than 95% purity and verified by HPLC and mass spectrometry. Biochemical properties of the peptides employed in this study are listed in Table 1.

Peptides were solubilised in d.H 2 0 at stock concentrations of 50 mg/mL. Caspofungin (Merck) and micafungin (Astellas) were diluted into 1 mg/mL and 10mg/mL aliquots with sterile d.H 2 O, respectively. Anidulafungin (Pfizer) was diluted into 10mg/mL aliquots with 20% v/v ethanol.

Checkerboard assay of yeast growth inhibition.

96-well microplate (Greiner Bio-one Ltd, Stonehouse, UK) wells were filled with 150µl RPMI-1640 (Sigma) with 2% MOPS (Sigma), pH 6.4. Appropriate concentrations of individual antifungals or combinations of peptides with the echinocandins were added. Approximately 500 cells from midexponential phase cultures of each yeast strain were then added to each well. Plates were incubated at 30ºC for 48 h and scanned using an ImageScanner (GE Healthcare UK Ltd, Chalfont St Giles, UK) with ImageMaster Labscan v 3.00 software (GE Healthcare UK Ltd). Images were annotated using Microsoft PhotoDraw. The MIC for each antifungal tested was defined as the concentration present in the first optically clear well after 48 h. Sterile paper discs (6mm, Aa, Whatman) were impregnated with the relevant peptide (5 to 25 μg) with or without echinocandin. These were left in sterile Petri dishes overnight at room temperature to dry. 100µl of mid-exponential phase cultures (OD 600 of 0.3) were spread onto RPMI-1640 agarose plates (2% agarose, 1% RPMI-1640 medium Auto-mod TM (Sigma), 0.4% sodium bicarbonate (Sigma), 2% MOPS (Sigma))and left to dry for 1 h at room temperature. The discs containing the various antifungal combinations were then applied to the surface of the plates and incubated at 30ºC for 48 h. Images showing the zones of inhibition were obtained by scanning the plates as described in section 2.3.

Assay of yeast cell viability.

Starter cultures were incubated overnight in MEB, pH 7 at 30 ºC with shaking. 1 mL of this culture was diluted with fresh MEB, pH 7 to give a starting OD 600 of 0.05 prior to incubation at 30ºC with shaking at 200 rpm. OD 600 readings were taken every 60 min until an OD 600 of 0.3 was reached.

Cultures were then exposed to peptide with or without caspofungin. Culture viability was measured every 30 min by serial dilution and plating onto YEPD agar plates. Plates were incubated at 30ºC for 48 h prior to enumeration of survivors.

In vitro mammalian cell cytotoxicity assay.

The method is as described in Yang et al. [30] and employed non-cancerous Vero cells (African green monkey kidney fibroblasts). Vero cells were exposed to echinocandins, antimicrobial peptides or combinations of both for 48 h and viability determined by the neutral red procedure.

Viable cells were expressed as the proportion retaining neutral red after exposure to antifungals relative to untreated controls. CCCP (Sigma) was used as a positive control (0.15 µg/mL).

Intracellular localisation of fluorescein tagged dermaseptin S3(1-16) using fluorescence microscopy.

The phase-contrast and fluorescent peptide images were generated using the FITC filter (Ex λ = 490/20 nm, Em λ = 528/38 nm). Propidium iodide (PI; Invitrogen) was used to identify dead cells was used for image processing and analysis.

Efficacy in vivo of the combination of caspofungin with ranalexin in a murine model of disseminated Candidiasis.

Basic details of the intravenous challenge model for disseminated C. albicans infection have been published previously [17]. Briefly, female BALB/c mice were infected intravenously with C. albicans at a dose of 3±1x10 4 CFU/g body weight. Animals were humanely terminated 72 h after challenge, and body weight changes, which correlate with survival times [16], and fungal burdens in kidney homogenates were determined.

Caspofungin IV preparation was diluted in saline as required. Combination therapy with caspofungin and ranalexin was investigated in two experiments. In the first, caspofungin was dosed at 0.01 mg/kg IP, qd, and ranalexin at 10 mg/kg IV, qd. Saline IV and IP was used as placebo, and animals given caspofungin or ranalexin monotherapy were concomitantly dosed with saline by the IV and IP routes, respectively. The first treatments were given 1 h after challenge and repeated at 24 and 48 h. In the second experiment, caspofungin was dosed at 0.05 mg/kg IV, qd, and ranalexin was dosed IV at 10 mg/kg bid. The first treatments were given 1 h after challenge.

Saline IV was used as placebo.

All animal experimentation was approved by the local ethical review committee and was performed under UK Home Office regulations. Statistical analysis of the data was by Mann-Whitney U-test.

Combination of caspofungin or anidulafungin with antimicrobial peptides results in potent, synergistic inhibition of C. albicans and C. glabrata in vitro.

Exposing C. albicans SC5314 to a combination of peptides with caspofungin (Figure 1), or anidulafungin (data not shown), resulted in significant enhancement of the inhibitory activity of the peptides when compared with peptide treatment alone. FICs calculated from the checkerboard assays (Table 2) showed that combination of all five peptides with caspofungin resulted in clear synergy (<0.5), particularly versus the C. albicans strains. Synergy against C. glabrata was evident to a lesser extent with all the peptides tested with the exception of ranalexin. The FIC values generated from combining anidulafungin with the same peptides revealed a similar pattern of synergy as occurred with caspofungin with the exception of cyclic peptide 6752 that displayed no synergy. As with caspofungin, the synergistic effect induced by combining the peptides with anidulafungin was most evident versus the C. albicans strains.

Similar data were also obtained from identical checkerboard assays using the hospital isolates of C. albicans and C. glabrata (data not shown). One peptide, gomesin, showed no enhanced inhibition of any of the strains tested in combination with any of the echinocandins (data not shown). Also, no enhancement of the inhibitory effect of all the peptides tested was observed when they were combined with micafungin (data not shown).

To determine if the synergistic inhibition of the combinations enhanced the fungicidal effect of the peptides or the echinocandins we tested the effect of the combination of caspofungin with ranalexin, magainin 2 and DsS3(1-16) on the viability of growing cells of C. albicans SC5314 (Figure 2). Addition of ranalexin (9 mg/L: Figure 2a), magainin 2 (10 mg/L: Figure 2b) or DsS3(1-16) (4 mg/L: Figure 2c) alone to exponentially growing cells had little effect on viability compared to untreated controls. In contrast, exposure to caspofungin alone (0.01 mg/L: Figure 2a, b andc) resulted in minor inhibition of growth rate. Notably, with cultures exposed to the individual peptides (at identical concentrations as described above), but in the presence of 0.01 mg/L caspofungin, in each case there was complete cessation of growth and notable loss of viability. Thus, the

In vitro toxicity testing of antimicrobials peptides alone and in combination with caspofungin.

In preparation for in vivo testing of the combination of antimicrobial peptides with caspofungin in a mouse model of systemic candidiasis, the in vitro toxicity of the antifungals on noncancerous Vero cells was evaluated (Figure 3). Exposure to caspofungin or anidulafungin resulted in a minor loss of cell viability at a concentration an order of magnitude higher than necessary to completely inhibit growth of C. albicans in vitro (Figure 3a). The antimicrobial peptides had no measurable toxic effect on Vero cells up to a concentration of 50 mg/L. However, exposure to 100 mg/L of the cyclic peptide GS24K4 and ranalexin resulted in approximately 70% and 20% loss of viability respectively (Figure 3b). Peptides 6752, DsS3(1-16) and magainin 2 induced no significant loss of cell viability even at the highest concentration tested: 100 mg/L. Following this, we tested the effect of combinations of either caspofungin (Figure 3c) or anidulafungin (Figure 3d) with each of the peptides. Combination of 10 mg/L caspofungin with 30 mg/L of any of the antimicrobial peptides had no detectable effect on Vero cell viability. However, caspofungin at 15 mg/L in combination with 50 mg/L of ranalexin showed some cytotoxicity and 100 mg/L caspofungin with 100 mg/L GS14K4, ranalexin and DsS3(1-16) induced significant loss of viability. Identical experiments testing the cytotoxicity of anidulafungin also showed that combination of 10 mg/L anidulafungin with 30 mg/L of any of the antimicrobial peptides had only a minor effect on Vero cell viability. At higher concentrations of anidulafungin and peptides toxicity increased significantly, demonstrating that peptide combinations with anidulafungin were potentially more cytotoxic than those with caspofungin. Nonetheless, the study revealed that concentrations of the antifungals in combination that are much higher than concentrations necessary to completely inhibit growth of C. albicans in vitro (Figure 1) did not induce significant cytotoxicity in vitro. Thus, these results indicated that there may be a therapeutic 'window' where the combination treatment may show efficacy versus C. albicans without inducing toxicity. Previous work with DsS3(1-16) tagged with fluorescein revealed that the peptide binds transiently to the outer cell wall prior to being taken up by the cell and sequestered into the vacuole before finally disseminating throughout the cytosol [12]. Thus, we used fluorescence microscopy to study how exposure to caspofungin affected the uptake of DsS3(1-16)-fluorescein. In addition to exposing the cells to DsS3(1-16)-fluorescein, we simultaneously stained with PI to correlate stages of peptide uptake with loss of viability (Figure 4). Exposure to 20 mg/L DsS3(1-16)-fluorescein for 2 hours resulted in approximately 5% of the population displaying fluorescence throughout the entire cell, 20% with fluorescence localised to the vacuole, and 75% of the population showing no peptide uptake (Figure 4a). As before [12], when the peptide was disseminated throughout the entire cell this correlated with PI uptake into the cells indicating that these cells were unviable (data not shown). In contrast, cells with the peptide concentrated inside the vacuole were not PI-positive and were viable (Figure 4b). Exposure to 0.001 mg/L caspofungin for 2 h resulted in approximately 15% of the population becoming PI-positive. However, if the cells were exposed to the caspofungin in the presence of DsS3(1-16)-fluorescein, the proportion of cells that were PI-positive increased to approximately 35% (Figure 4a). As before, all these PI-positive cells also displayed green fluorescence throughout the entire cell indicating complete peptide entry into the cells. Exposure to 0.01 or 0.05 mg/L caspofungin alone resulted in approximately 24 and 26%, respectively, of the population becoming PI-positive after 2 h (Figure 4a). Paradoxically, at higher concentrations of caspofungin, 0.1 and 1 mg/L respectively, the proportion of cells that were PIpositive decreased to approximately 19% in each case. This phenomenon was seen in many repeat experiments with both caspofungin and anidulafungin (data not shown). In identical experiments using anidulafungin instead of caspofungin, similar trends with regard to peptide uptake were observed (data not shown).

Upon addition of

Combination of caspofungin with ranalexin did not show synergistic inhibition of C. albicans in a murine model of disseminated Candidiasis.

The results of the mouse experiments are summarized in Table 3. In the first experiment, with caspofungin given IP at 0.01 mg/kg, the 72 h kidney burden was reduced significantly below placebo (p<0.05) in mice given the caspofungin/ranalexin combination: however, the extent of the effect was less than a 1-log burden reduction. No significant reduction in weight loss relative to placebo-treated mice was seen for any of the test agents. In the second experiment, with a higher caspofungin dose of 0.05 mg/kg, IV dosing of both test agents, and BID dosing of ranalexin, highly significant (p<0.01) reductions in kidney burdens and 3-day weight loss were seen for groups of animals receiving treatments that included caspofungin. However, progressive necrosis of the tails of animals given ranalexin alone or in combination meant that treatments with this agent were stopped after 36 h in the interests of animal welfare.

Combination of antimicrobial peptides with caspofungin results in potent, synergistic killing of C. albicans on a porous, solid surface.

To explore whether the combination treatment displayed synergy in a topical application, lawns of C. albicans SC5314 (Figure 5a) and the C. albicans hospital isolate (Figure 5b) covering the surface of RPMI1640 agarose plates were exposed to sterile, filter paper discs impregnated with caspofungin, ranalexin, magainin 2 or DsS3(1-16) alone and combinations of caspofungin with each peptide. With both strains, caspofungin impregnated discs showed only minor zones of inhibition at the higher concentrations tested of 0.075, 0.1 and 0.124 μg. Exposure of both strains A c c e p t e d M a n u s c r i p t 13 to discs impregnated with each peptide alone up to the highest concentration of 25 μg resulted in no detectable zones of inhibition. However, exposure of both strains to discs impregnated with combinations of each peptide with caspofungin clearly revealed synergistic inhibition, with larger, more significant zones of inhibition after 48 h exposure. Similar results were observed for both yeast strains exposed to combinations of the same peptides with anidulafungin (data not shown).

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

Discussion

Combination therapy using clinically-approved antifungals is commonly employed to treat mycotic disease (reviewed by Mukherjee et al. [19]) eg., combinations of intravenous amphotericin B or oral fluconazole with 5-fluorocytosine have been proposed for treatment of candidiasis [22]. Synergy between caspofungin and amphotericin B against Aspergillus spp. has been descibed in vitro [1],

and in a murine model [15], and resulted in improved therapeutic outcome in a murine model of systemic C. glabrata infection [21]. However, controversy surrounds whether or not combination therapy is superior to monotherapy in clinical situations due to a lack of evidence from clinical trials [4].

There are examples in the literature of apparent synergy, or enhanced efficacy, between various antimicrobial peptides and clinically-approved antifungals eg., the combination of Peptide 2, a derivative of lactoferrin, with amphotericin B resulted in enhanced survival of mice in a model of systemic candidiasis [25]. Notably, there are no published reports of synergistic interactions between antimicrobial peptides and the clinically-approved echinocandins.

Despite the introduction of new antifungals, such as voriconazole and caspofungin, in some cases antifungal efficacies remain sub-optimal [5] and there is increasing evidence of antifungal therapy failing due to the selection in vivo of resistant fungal pathogens eg., reduced susceptibility to caspofungin in patients receiving therapy for invasive candidiasis [26]. Thus, the use of combinations of antifungals in therapy could be an effective alternative approach to treat invasive fungal infections that are not responsive to standard monotherapy.

Here, we describe how combination of the echinocandins, caspofungin and anidulafungin, with antimicrobial peptides results in significant synergy against Candida spp. in vitro. Notably, we detected no apparent synergy using identical combinations of peptides with the other clinicallyapproved echinocandin, micafungin. This is surprising considering the similarity in structure, and apparent inhibitory action, between all of these drugs and merits further investigation. The FIC values calculated from the checkerboard experiments revealed that all the antimicrobial peptides tested (with the exception of the cyclic peptide 6752 in combination with anidulafungin) displayed A c c e p t e d M a n u s c r i p t some degree of synergy against the three yeasts tested in combination with either caspofungin or anidulafungin (Table 2). With each combination tested, the level of synergy against the hospitalisolate of C. glabrata was reduced compared to that shown against the two C. albicans strains. This is not surprising considering C. glabrata has been shown to show greater resistance than C. albicans to many antifungal drugs, particularly the triazoles [23].

Study of the nature of the inhibitory mechanism of the synergistic combination using fluorescence microscopy revealed that exposure to the cell-wall degrading effect of caspofungin lead to enhanced uptake of the cationic peptide into the cell, resulting in enhanced killing. Intriguingly, there was evidence of paradoxical resistance to increasing concentrations of caspofungin as the degree of peptide uptake into the cells initially increased with higher concentrations but then declined again as the concentration was raised further. Evidence of a paradoxical resistance phenotype to echinocandins has been presented before with Candida spp., particularly with those isolates exposed to caspofungin [2]. Possible explanations for this paradoxical effect include; the induction of stress-response pathways [28], or, the elevation in the chitin content of the cell walls of C. albicans cells exposed to caspofungin reducing the penetration of the peptide into the cells [27].

Ideally, whether or not a combination treatment could prove efficacious in vivo would be assessed using animal models followed by clinical trials. Thus, we employed a murine model of disseminated candidasis to determine if the combination treatment demonstrated enhanced efficacy in vivo. We chose to use caspofungin and ranalexin due to previous experience of in vivo dosing with these two compounds. Disappointingly, the combination treatment did not result in enhanced efficacy compared to the single treatments. However, resources only permitted a preliminary experiment with only one dose of each of the compounds tested and we were only able to test the in vivo efficacy of one echinocandin, caspofungin, with one peptide, ranalexin. As the experiment progressed there was evidence of ranalexin toxicity which was manifest as tail vein necrosis and ulceration at the site(s) of administration. Clearly, the pharmacokinetics and interactions of antifungal peptides, where little is known, is difficult to optimise in an animal model. Furthermore, it is even more difficult to replicate conditions present in humans. Nonetheless, there is additional scope to use the mouse model to study the effects of alternative, non-toxic peptides in combination A c c e p t e d M a n u s c r i p t 16 with other echinocandins, with varied dosing regimens and alternative routes of administration, versus additional fungal pathogens such as Aspergillus spp.. Thus, despite this preliminary failure, we believe that a combination of an echinocandin with an antimicrobial peptide can be identified that could still result in enhanced efficacy in humans. Importantly, we have demonstrated synergistic inhibition of yeast growing on the surface of an agarose plate and if intravenous administration of the combination treatment is not efficacious then topical administration to treat conditions such as oral or mucosal candidiasis could prove to be more effective. A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t Fungicidal synergy was measured by calculating fractional inhibitory concentration indices from checkerboard assays as well as loss of viability. Inhibitory combinations of the antifungals did not induce cytotoxicity in vitro. However, in a murine model of systemic candidiasis, co-administration of caspofungin with one example of the cationic peptides tested, ranalexin, did not show enhanced efficacy compared with the single treatments alone. Further study using alternative peptides will identify whether this combination approach could represent a novel treatment for fungal pathogens.

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

Introduction

Over the past 30 years, fungi have emerged as significant causes of human disease, with attendant morbidity and mortality [1,2]. A large proportion of these infections occur in immunocompromised patients, as fungal pathogens are readily able to overwhelm the already weakened host defence mechanisms. Hospitalised patients with serious underlying diseases are increasingly exposed to invasive surgical procedures or chemotherapeutic treatments, such as broad-spectrum antibiotics or immunosuppressants following organ transplantation and cancer chemotherapy, that readily promote mycotic disease. Infections caused by Candida albicans have a population incidence of between 1.1 and 24 cases per 100 000 individuals with an associated mortality rate of >30% [3,4].

In the USA this equates to between 3000 and 11 000 deaths annually from nosocomial candidaemia [5]. Notably, treatment with many antifungal drugs is complicated by high toxicity and low tolerability, ineffectiveness against new or re-emerging fungi, or the development of drug-resistant strains in patients undergoing treatment [5]. Thus, there is a justifiable need for more research to develop new antifungal treatments.

One possible approach to improve the range and scope of antifungal therapy is the use of combinations of antifungals. Benefits could include: improved efficacy over the use of single drugs, consequently allowing lower, less costly doses of each individual drug; reduced toxicity and improved tolerance; broader spectrum of efficacy; and finally, the potential to prevent resistant organisms arising as can occur with monotherapy.

A potential source of new antifungals is the cationic antimicrobial peptides (AMPs), which are ubiquitous throughout nature and protect against invasive microbial pathogens. To date, more than 800 AMPs have been described [6]. Potential advantages of cationic synthesising and producing large quantities of these molecules [7]. Employing cationic peptides in combination with other clinically approved drugs could overcome some of the problems associated with the clinical application of peptides.

Echinocandins inhibit the synthesis of -D-glucan in fungal cell walls and are fungicidal against Candida spp. and fungistatic against Aspergillus spp. owing to loss of cell wall integrity [8]. Importantly, an intact cell wall also represents a potential barrier that cationic peptides must interact with and pass through before they can interact with the plasma membrane beneath. Supporting this, recent data from our laboratory have demonstrated that disruption of the outer cell wall phosphomannan content has a significant influence on the degree of susceptibility of C. albicans to a cationic peptide [9]. Thus, owing to the complementary inhibitory action of the echinocandins and cationic peptides, we reasoned that application of combinations of these molecules could result in additive or synergistic inhibition of fungal pathogens. In this study, we combined the three clinically approved echinocandins with a range of structurally diverse AMPs and observed potent synergistic killing of Candida spp. in vitro.

A c c e p t e d M a n u s c r i p t 5

Material and methods

Yeast strains and growth media

Yeast strains used in this work were C. albicans SC5314 and clinical isolates of C.

albicans and Candida glabrata provided by Dr Cyril Lafong (Fife Area Laboratory, Victoria Hospital, Kirkcaldy, UK). All strains were cultured in malt extract broth (MEB) (pH 7) (1% glucose, 0.6% malt extract and 0.12% yeast extract) at 30 C with shaking. To enumerate cell survival after exposure to antifungals, yeasts were plated on YEPD agar (2% glucose, 2% agar, 1% Bacto Peptone and 1% yeast extract). Numbers of cells used in the various assays described below were calculated using an optical density at 600 nm (OD 600 ) versus a cell number calibration curve for each yeast strain used (data not shown).

Antifungals

Magainin 2 [10], ranalexin [11], cyclic peptide 6752 [12], cyclic peptide GS14K4 [13],

gomesin [14] PhotoDraw. The minimum inhibitory concentration for each antifungal tested was defined as the concentration present in the first optically clear well after 48 h.

Disk diffusion assay of yeast growth inhibition

In vitro mammalian cell cytotoxicity assay

The method for the in vitro mammalian cell cytotoxicity assay is as described by Yang et al. [16] and employed non-cancerous Vero cells (African green monkey kidney fibroblasts).

Vero cells were exposed to echinocandins, AMPs or combinations of both for 48 h and viability was determined by the neutral red procedure. Viable cells were expressed as the proportion retaining neutral red after exposure to antifungals relative to untreated controls.

Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) 0.15 g/mL (Sigma) was used as a positive control.

Intracellular localisation of fluorescein-tagged dermaseptin S3(1-16) using fluorescence microscopy

Phase contrast and fluorescent peptide images were generated using the fluorescein

In vivo efficacy of the combination of caspofungin with ranalexin in a murine model of disseminated candidiasis

Basic details of the intravenous (i.v.) challenge model for disseminated C. albicans infection have been published previously [17]. Briefly, female BALB/c mice were infected intravenously with C. albicans at a dose of 3 ± 1  10 4 colony-forming units/g body weight.

Animals were humanely terminated 72 h after challenge and body weight changes, which correlate with survival times [18], as well as fungal burdens in kidney homogenates were determined.

Caspofungin i.v. preparation was diluted in saline as required. Combination therapy with caspofungin and ranalexin was investigated in two experiments. In the first experiment, caspofungin was dosed at 0.01 mg/kg via the intraperitoneal (i.p.) route once daily and ranalexin was dosed at 10 mg/kg i.v. once daily. Saline i.v. and i.p. was used as placebo, and animals given caspofungin or ranalexin monotherapy were concomitantly dosed with A c c e p t e d M a n u s c r i p t 9 saline by the i.v. and i.p. routes, respectively. The first treatments were given 1 h after challenge and treatments were repeated at 24 h and 48 h. In the second experiment, caspofungin was dosed at 0.05 mg/kg i.v. once daily and ranalexin was dosed at 10 mg/kg i.v. twice daily. The first treatments were given 1 h after challenge. Saline i.v. was used as placebo.

Statistical analysis of the data was performed by the Mann-Whitney U-test.

Results

Combination of caspofungin or anidulafungin with antimicrobial peptides results in potent synergistic inhibition of Candida albicans and Candida glabrata in vitro

Exposing C. albicans SC5314 to a combination of peptides with caspofungin (Fig. 1) or anidulafungin (data not shown) resulted in significant enhancement of the inhibitory activity of the peptides compared with peptide treatment alone. Similar data were also obtained from identical checkerboard assays using the hospital isolates of C. albicans and C. glabrata (data not shown). Fractional inhibitory concentration indices (FICIs) calculated from the checkerboard assays (Table 2) showed that combination of all five peptides with caspofungin resulted in clear synergy (<0.5), particularly versus the C. albicans strains.

Synergy against C. glabrata was evident to a lesser extent with all the peptides tested with the exception of ranalexin. The FICI values generated from combining anidulafungin with the same peptides revealed a similar pattern of synergy as occurred with caspofungin, with the exception of cyclic peptide 6752 that displayed no synergy. As with caspofungin, the synergistic effect induced by combining the peptides with anidulafungin was most evident against the C. albicans strains.

A c c e p t e d M a n u s c r i p t 10 One peptide, gomesin, showed no enhancement of inhibition of any of the strains tested in combination with any of the echinocandins (data not shown). Also, no enhancement of the inhibitory effect of all the peptides tested was observed when they were combined with micafungin (data not shown).

To determine whether the synergistic inhibition of the combinations enhanced the fungicidal effect of the peptides or the echinocandins, the effect of the combination of caspofungin with ranalexin, magainin 2 and DsS3(1-16) on the viability of growing cells of C. albicans SC5314 was tested (Fig. 2). Addition of 9 mg/L ranalexin (Fig. 2a), 10 mg/L magainin 2 (Fig. 2b) or 4 mg/L DsS3(1-16) (Fig. 2c) alone to exponentially growing cells had little effect on viability compared with untreated controls. In contrast, exposure to caspofungin alone at 0.01 mg/L (Fig. 2a-c) resulted in minor inhibition of the growth rate.

Notably, with cultures exposed to the individual peptides (at identical concentrations as described above) but in the presence of 0.01 mg/L caspofungin, in each case there was complete cessation of growth and notable loss of viability. Thus, the combination of AMPs with either caspofungin or anidulafungin resulted in potent synergistic fungicidal activity.

In vitro toxicity testing of antimicrobials peptides alone and in combination with caspofungin

In preparation for in vivo testing of the combination of AMPs with caspofungin in a mouse model of systemic candidiasis, the in vitro toxicity of the antifungals on non-cancerous Vero cells was evaluated (Fig. 3). Exposure to caspofungin or anidulafungin resulted in a minor loss of cell viability at a concentration an order of magnitude higher than necessary to completely inhibit growth of C. albicans in vitro (Fig. 3a). The AMPs had no measurable toxic effect on Vero cells up to a concentration of 50 mg/L. However, exposure to 100 mg/L of the cyclic peptide GS14K4 and ranalexin resulted in ca. 70% and 20% loss of Following this, the effect of combinations of either caspofungin (Fig. 3c) or anidulafungin (Fig. 3d) with each of the peptides was tested. Combination of 10 mg/L caspofungin with 30 mg/L of any of the AMPs had no detectable effect on Vero cell viability. However, 15 mg/L caspofungin in combination with 50 mg/L ranalexin showed some cytotoxicity, and 100 mg/L caspofungin with 100 mg/L GS14K4, ranalexin and DsS3(1-16) induced significant loss of viability. Identical experiments testing the cytotoxicity of anidulafungin also showed that combination of 10 mg/L anidulafungin with 30 mg/L of any of the AMPs had only a minor effect on Vero cell viability. At higher concentrations of anidulafungin and peptides, toxicity increased significantly, demonstrating that peptide combinations with anidulafungin were potentially more cytotoxic than those with caspofungin. None the less, the study revealed that concentrations of antifungals in combination that are much higher than concentrations necessary to completely inhibit growth of C. albicans in vitro (Fig. 1)

did not induce significant cytotoxicity in vitro. Thus, these results indicated that there may be a therapeutic 'window' where the combination treatment may show efficacy versus C.

albicans without inducing toxicity. were simultaneously stained with PI to correlate stages of peptide uptake with loss of viability (Fig. 4). Exposure to 20 mg/L DsS3(1-16)-fluorescein for 2 h resulted in ca. 5% of the population displaying fluorescence throughout the entire cell, 20% with fluorescence localised to the vacuole and 75% of the population showing no peptide uptake (Fig. 4a).

As reported previously [9], when the peptide was disseminated throughout the entire cell this correlated with PI uptake into the cells, indicating that these cells were non-viable (data not shown). In contrast, cells with the peptide concentrated inside the vacuole were not PI-positive and were viable (Fig. 4b).

Exposure to 0.001 mg/L caspofungin for 2 h resulted in ca. 15% of the population becoming PI-positive. However, if the cells were exposed to caspofungin in the presence of DsS3(1-16)-fluorescein, the proportion of cells that were PI-positive increased to ca. 35% (Fig. 4a). As before, all these PI-positive cells also displayed green fluorescence throughout the entire cell indicating complete peptide entry into the cells.

Exposure to 0.01 mg/L or 0.05 mg/L caspofungin alone resulted in ca. 24% and 26%, respectively, of the population becoming PI-positive after 2 h (Fig. 4a). Paradoxically, at higher concentrations of caspofungin (0.1 mg/L and 1 mg/L), the proportion of cells that were PI-positive decreased to ca. 19% in each case. This phenomenon was seen in many repeat experiments both with caspofungin and anidulafungin (data not shown). In identical experiments using anidulafungin instead of caspofungin, similar trends with regard to peptide uptake were observed (data not shown).

Upon addition of

Combination of caspofungin with ranalexin did not show synergistic inhibition of Candida albicans in a murine model of disseminated candidiasis

Results of the mouse experiments are summarised in Table 3. In the first experiment with caspofungin given i.p. at 0.01 mg/kg, the 72 h kidney burden was reduced significantly below placebo (P < 0.05) in mice given the caspofungin/ranalexin combination; however, the extent of the effect was <1 log burden reduction. No significant reduction in weight loss relative to placebo-treated mice was seen for any of the test agents. In the second experiment with a higher caspofungin dose of 0.05 mg/kg, i.v. dosing of both test agents and twice daily dosing of ranalexin, highly significant (P < 0.01) reductions in kidney burdens and 3-day weight loss were seen for groups of animals receiving treatments that included caspofungin. However, progressive necrosis of the tails of animals given ranalexin alone or in combination meant that treatments with this agent were stopped after 36 h in the interests of animal welfare. To explore whether the combination treatment displayed synergy in a topical application, lawns of C. albicans SC5314 (Fig. 5a) and the C. albicans hospital isolate (Fig. 5b) covering the surface of RPMI 1640 agarose plates were exposed to sterile filter paper disks impregnated with caspofungin, ranalexin, magainin 2 or DsS3(1-16) alone as well as combinations of caspofungin with each peptide. For both strains, caspofungin-impregnated disks showed only minor zones of inhibition at the higher concentrations tested of 0.075, 0.1 and 0.124 g. Exposure of both strains to disks impregnated with each peptide alone up to the highest concentration of 25 g resulted in no detectable zones of inhibition.

However, exposure of both strains to disks impregnated with combinations of each peptide with caspofungin clearly revealed synergistic inhibition, with larger, more significant zones of inhibition after 48 h exposure. Similar results were observed for both yeast strains exposed to combinations of the same peptides with anidulafungin (data not shown).

Discussion

Combination therapy using clinically approved antifungals is commonly employed to treat mycotic disease (reviewed by Mukherjee et al. [20]), e.g. combinations of i.v. amphotericin B or oral fluconazole with 5-fluorocytosine have been proposed for the treatment of candidiasis [21]. Synergy between caspofungin and amphotericin B against Aspergillus spp. has been described in vitro [22] and in a murine model [23] and resulted in improved therapeutic outcome in a murine model of systemic C. glabrata infection [24]. However, controversy surrounds whether or not combination therapy is superior to monotherapy in clinical situations owing to a lack of evidence from clinical trials [25]. Despite the introduction of new antifungals such as voriconazole and caspofungin, in some cases antifungal efficacies remain suboptimal [27] and there is increasing evidence of antifungal therapy failing due to the selection in vivo of resistant fungal pathogens, e.g. reduced susceptibility to caspofungin in patients receiving therapy for invasive candidiasis [28]. Thus, the use of combinations of antifungals in therapy could be an effective alternative approach to treat invasive fungal infections that are not responsive to standard monotherapy.

Here we describe how combination of the echinocandins caspofungin and anidulafungin with AMPs results in significant synergy against Candida spp. in vitro. Notably, no apparent synergy was detected using identical combinations of peptides with the other clinically approved echinocandin, micafungin. This is surprising considering the similarity in structure and apparent inhibitory action between all of these drugs and merits further investigation. The FICI values calculated from the checkerboard experiments revealed that all the AMPs tested (with the exception of the cyclic peptide 6752 in combination with anidulafungin) displayed some degree of synergy against the three yeasts tested in combination with either caspofungin or anidulafungin (Table 2). With each combination tested, the level of synergy against the hospital isolate of C. glabrata was reduced compared with that shown against the two C. albicans strains. This is not surprising to many antifungal drugs, particularly the triazoles [5].

Study of the nature of the inhibitory mechanism of the synergistic combination using fluorescence microscopy revealed that exposure to the cell wall-degrading effect of caspofungin led to enhanced uptake of the cationic peptide into the cell, resulting in enhanced killing. Intriguingly, there was evidence of paradoxical resistance to increasing concentrations of caspofungin, as the degree of peptide uptake into the cells initially increased with higher concentrations but then declined again as the concentration was raised further. Evidence of a paradoxical resistance phenotype to echinocandins has been presented previously for Candida spp., particularly with those isolates exposed to caspofungin [29]. Possible explanations for this paradoxical effect include the induction of stress response pathways [30] or elevation in the chitin content of the cell walls of C.

albicans cells exposed to caspofungin, reducing penetration of the peptide into the cells [31].

Ideally, whether or not a combination treatment could prove efficacious in vivo would be assessed using animal models followed by clinical trials. Thus, a murine model of disseminated candidiasis was employed to determine whether the combination treatment demonstrated enhanced efficacy in vivo. Caspofungin and ranalexin were chosen owing to previous experience of in vivo dosing with these two compounds. Disappointingly, the combination treatment did not result in enhanced efficacy compared with the single treatments. However, resources only permitted a preliminary experiment with only one dose of each of the compounds tested and it was only possible to test the in vivo efficacy of one echinocandin, caspofungin, with one peptide, ranalexin. As the experiment progressed there was evidence of ranalexin toxicity, which was manifest as tail vein necrosis and ulceration at the site(s) of administration. Clearly, the pharmacokinetics and interactions of antifungal peptides, about which little is known, are difficult to optimise in an animal model. Furthermore, it is even more difficult to replicate conditions present in humans. None the less, there is additional scope to use the mouse model to study the effects of alternative non-toxic peptides in combination with other echinocandins, with varied dosing regimens and alternative routes of administration, against additional fungal pathogens such as Aspergillus spp. Thus, despite this preliminary failure, we believe that a combination of an echinocandin with an AMP can be identified that could still result in enhanced efficacy in humans. Importantly, we have demonstrated synergistic inhibition of yeast growing on the surface of an agarose plate, and if i.v. administration of the combination treatment is not efficacious then topical administration to treat conditions such as oral or mucosal candidiasis could prove to be more effective. b FICI values in bold indicate combinations where no synergy was observed.

Edited Table 2

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

 4 with other clinically-approved drugs could overcome some of the problems associated with the clinical application of peptides.

A c c e p t e d M a n u s c r i p t 6 2. 4 .

 64 Disc diffusion assay of yeast growth inhibition.

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

 7 RD-TR-PE, Ex λ = 490/20 nm, Em λ = 528/38 nm). Cells were cultured in MEB and harvested at mid exponential phase (OD 600 0.6). A volume equivalent to 2.0 x10 6 cells/ml was removed for each assay, centrifuged for 2 min at 10 000 rpm, washed in ddH 2 O and resuspended in 1 ml RPMI-1640. Echinocandin and/or DsS3(1-16)-fluorescein were added with PI (1.8 µM) and incubated for 2 h at 30 o C in the dark. Unbound dye and peptide were removed by harvesting for 8 min at 3 000 x g, washing the pellet in ddH 2 O, harvesting and resuspension in 20 µl RPMI-1640. 2 µl of the stained cells were fixed with 2 µl of 1 % low-melting-point agarose (Biogene). Samples were placed on ice and protected from light until analysis. Images were captured on an Olympus IX70 DeltaVision microscope (Applied Precision LLC). SoftWoRx Explorer 1.3 (Applied Precision LLC)

A c c e p t e d M a n u s c r i p t 10 combination

 10 of antimicrobial peptides with either caspofungin or anidulafungin resulted in potent, synergistic fungicidal activity.

11

 11

3. 3

 3 Exposure to an antimicrobial peptide in combination with caspofungin results in increased intracellular accumulation of the peptide and enhanced killing of C. albicans.

  DsS3(1-16)-fluorescein to increasing concentrations of caspofungin (0.01 -1 mg/L) we observed a similar trend to that observed previously with an increase in the proportion of cells in the population becoming PI-positive and simultaneously displaying total green fluorescence throughout the cell. Coincident with the increase in the proportion of cells displaying total dissemination of DsS3(1-16)-fluorescein throughout the entire cell, at every concentration of A c c e p t e d M a n u s c r i p t caspofungin tested, addition of DsS3(1-16)-fluorescein resulted in a smaller proportion of the population displaying only vacuolar localisation of the peptide. Thus, the presence of caspofungin results in greater, or accelerated, uptake of the peptide into the cytosol, which in turn, leads to enhanced loss of cell viability.

  Figure 1. -Checkerboard growth assay(s) of; antimicrobial peptide MIC's alone, and each of the

  Figure 1. -Checkerboard growth assay(s) of; antimicrobial peptide MIC's alone, and each of the

Figure 2 .

 2 Figure 2. -The effect of combination of antimicrobial peptides with caspofungin on the growth and viability of C. albicans SC5314. Cultures were grown in MEB, pH 7 at 30 o C with shaking and

Figure 3 .

 3 Figure 3. -In vitro toxicity of the echinocandins, antimicrobial peptides and combinations of

Figure 4 .

 4 Figure 4. -Microscopic study on how the presence of caspofungin effects the uptake of DsS3(1-16)-fluorescein into C. albicans SC5314. a) Yeast cells in RPMI-1640, supplemented with 1.8 μM PI, were exposed to the following conditions for 2 h at 30 o C: DsS3(1-16)-fluorescein (20 mg/L) alone; increasing concentrations of caspofungin (0.001 -1 mg/L); or, combinations of DsS3(1-16)fluorescein (20 mg/L) with increasing concentrations of caspofungin (0.001 -1 mg/L).

Figure 5 .

 5 Figure 5. -The effect of antimicrobial peptides and/or caspofungin impregnated into filter paper

Figure1

  Figure1Click here to download high resolution image

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

 2 Administering synergistic combinations of antifungals could be a route to overcome problems with toxicity and the development of resistance. Combination of the echinocandins caspofungin or anidulafungin with a range of structurally diverse antimicrobial peptides resulted in potent synergistic killing of Candida spp. in vitro.

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

 4 for future therapy include: a broad spectrum of activity; rapid killing of microbes; they are unaffected by classical antibiotic resistance mechanisms; they can neutralise endotoxin; and they are active in animal models[6]. Disadvantages include: sensitivity to proteolytic degradation by serum proteases in vivo; potential toxicity at high doses necessary to show efficacy in vivo, including haemolytic activity; and the high cost of

A c c e p t e d M a n u s c r i p t 7 2. 5 .

 75 Sterile paper disks (6 mm; Whatman International Ltd. Maidstone, UK) were impregnated with the relevant peptide (5-25 g) with or without echinocandin. Disks were left in sterile Petri dishes overnight at room temperature to dry. Then, 100 L of mid exponential phase cultures (OD 600 = 0.3) were spread onto RPMI 1640 agarose plates [2% agarose, 1% RPMI 1640 medium Auto-Mod TM (Sigma), 0.4% sodium bicarbonate (Sigma), 2% MOPS (Sigma)] and left to dry for 1 h at room temperature. Disks containing the various antifungal combinations were then applied to the surface of the plates and incubated at 30 C for 48 h. Images showing the zones of inhibition were obtained by scanning the plates as described in Section 2.3. Assay of yeast cell viabilityStarter cultures were incubated overnight in MEB (pH 7) at 30 C with shaking. Then, 1 mL of this culture was diluted with fresh MEB (pH 7) to give a starting OD 600 of 0.05 prior to incubation at 30 C with shaking at 200 rpm. OD 600 readings were taken every 60 min until an OD 600 of 0.3 was reached. Cultures were then exposed to peptide with or without caspofungin. Culture viability was measured every 30 min by serial dilution and plating onto YEPD agar plates. Plates were incubated at 30 C for 48 h prior to enumeration of survivors.

  isothiocyanate filter (Ex  = 490/20 nm, Em  = 528/38 nm). Propidium iodide (PI) (Invitrogen, Paisley, UK) was used to identify dead cells (RD-TR-PE, Ex  = 490/20 nm, Em  = 528/38 nm). Cells were cultured in MEB and were harvested at mid exponentialA c c e p t e d M a n u s c r i p t 8 phase (OD 600 = 0.6). A volume equivalent to 2.0  10 6 cells/mL was removed for each assay, centrifuged for 2 min at 10 000 rpm, washed in ddH 2 O and re-suspended in 1 mL of RPMI 1640. Echinocandin and/or DsS3(1-16)-fluorescein were added with PI (1.8 M) and incubated for 2 h at 30 C in the dark. Unbound dye and peptide were removed by harvesting for 8 min at 3000  g, washing the pellet in ddH 2 O, harvesting and resuspension in 20 L of RPMI 1640. Then, 2 L of the stained cells were fixed with 2 L of 1% low-melting-point agarose (BioGene, Huntingdon, UK). Samples were placed on ice and protected from light until analysis. Images were captured on an Olympus IX70 DeltaVision microscope (Applied Precision LLC, Washington, DC). SoftWoRx ® Explorer 1.3 (Applied Precision LLC) was used for image processing and analysis.

A c c e p t e d M a n u s c r i p t 11 viability

 11 , respectively (Fig.3b). Peptides 6752, DsS3(1-16) and magainin 2 induced no significant loss of cell viability even at the highest concentration tested (100 mg/L).

3. 3 .

 3 Exposure to an antimicrobial peptide in combination with caspofungin results in increased intracellular accumulation of the peptide and enhanced killing of Candida albicans Previous work with DsS3(1-16) tagged with fluorescein revealed that the peptide binds transiently to the outer cell wall prior to being taken up by the cell and sequestered into the vacuole before finally disseminating throughout the cytosol [9]. Thus, fluorescence microscopy was used to study how exposure to caspofungin affected the uptake of DsS3(1-16)-fluorescein. In addition to exposing cells to DsS3(1-16)-fluorescein, cellsA c c e p t e d M a n u s c r i p t 12

  DsS3(1-16)-fluorescein to increasing concentrations of caspofungin (0.01-1 mg/L), a similar trend to that observed previously was observed, with an increase in the proportion of cells in the population becoming PI-positive and simultaneously displaying total green fluorescence throughout the cell. Coincident with the increase in the proportion of cells displaying total dissemination of DsS3(1-16)-fluorescein throughout the A c c e p t e d M a n u s c r i p t 13 entire cell, at every concentration of caspofungin tested addition of DsS3(1-16)-fluorescein resulted in a smaller proportion of the population displaying only vacuolar localisation of the peptide. Thus, the presence of caspofungin results in greater, or accelerated, uptake of the peptide into the cytosol, which in turn leads to enhanced loss of cell viability.

A c c e p t e d M a n u s c r i p t 14 3. 5 .

 145 Combination of antimicrobial peptides with caspofungin results in potent synergistic killing of Candida albicans on a porous solid surface

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

 15 are examples in the literature of apparent synergy or enhanced efficacy between various AMPs and clinically approved antifungals, e.g. the combination of peptide 2, a derivative of lactoferrin, with amphotericin B resulted in enhanced survival of mice in a model of systemic candidiasis[26]. Notably, there are no published reports of synergistic interactions between AMPs and the clinically approved echinocandins.

A c c e p t e d M a n u s c r i p t 16 considering

 16 that C. glabrata has been shown to show greater resistance than C. albicans

Fig. 1 .

 1 Fig. 1. Checkerboard growth assays of minimum inhibitory concentrations of each

Fig. 2 .

 2 Fig. 2. Effect of the combination of antimicrobial peptides (AMPs) with caspofungin (0.01

Fig. 3 .

 3 Fig. 3. In vitro toxicity of the echinocandins, the antimicrobial peptides (AMPs) and

Fig. 4 .

 4 Fig. 4. Microscopic study of the effect of the presence of caspofungin on uptake of

Fig. 5 .

 5 Fig. 5. Effect of antimicrobial peptides (AMPs) and/or caspofungin impregnated into filter

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

 1

Table 1 .

 1 -Biochemical properties of the antimicrobial peptides used in this study. In addition to the listed references, data was also obtained from the Antimicrobial Peptide Database (http://aps.unmc.edu/AP/main.php ).Amino acid residues underlined represent D amino acids.

	Peptide	Sequence a	Length	Structure	Net	%	100%	Reference
					Charge	Hydrophobic	haemolysis
						residues	of RBCs
	Dermaseptin	ALWKNMLKGIGKLAGK	16	Helix	+4	50	>150 μM	[18]
	S3(1-16)
	Magainin 2	GIGKFLHSAKKFGKAFVGEIMNS	23	Helix	+4	43	>150 μg/ml	[31]
	Ranalexin	FLGGLIKIVPAMICAVTKKC	20	Helix with	+3	65	>500 μg/ml	[3]
				heptapeptide
				loop from 1
				disulphide
				bridge
	6752	SWFKTKSK	8	Cyclic	+3	25	>400 μg/ml	[6]
	(synthetic)
	GS14K4	VKLKVYPLKVKLYP	14	Cyclic	+4	42	200 μg/ml	[14]
	Gomesin	ECRRLCYKQRCVTYCRGR	18	β-hairpin	+6	33	Approx.	[24]
				with 2			20%
				disulphide			haemolysis
				bridges			at 100μM

a

Table 1

 1

Table 2 .

 2 -Fractional inhibitory concentration (FIC) indices for combinations of caspofungin or anidulafungin with various antifungal peptides calculated from chequerboard assays of visible growth inhibition of three Candida strains. FICs were calculated according to Odds[20] where values <0.5 indicate synergy and 0.5 to 4 no interaction. FIC values highlighted in grey indicate combinations where no synergy was observed.

			Caspofungin			Anidulafungin
	Peptide	C. albicans	C. albicans	C. glabrata	C. albicans	C. albicans	C. glabrata
		SC5314	(Hospital	(Hospital	SC5314	(Hospital	(Hospital
			isolate)	isolate)		isolate)	isolate)
	DsS3(1-16)	0.164	0.164	0.282	0.107	0.111	0.287
	Ranalexin	0.221	0.164	0.186	0.106	0.111	0.443
	Magainin 2	0.144	0.135	0.278	0.095	0.087	0.376
	6752	0.150	0.289	0.347	0.773	0.563	0.737
	GS14K4	0.267	0.139	0.269	0.142	0.287	0.341

Table 2

 2

Table 3 .

 3 -The effect of combination treatment with caspofungin and ranalexin compared to the individual treatments alone on kidney burden of C. albicans SC5314 and animal weight change in a mouse model of disseminated candidiasis.

	Kidney log 10	Weight change
	CFU/g	(%) day 0-day 3

*p<0.05 and **p<0.01 relative to placebo-treated group (Mann-Whitney U test).

Table 3

 3

Table 1

 1 Biochemical properties of the antimicrobial peptides used in this study Amino acid residues underlined represent D amino acids.

	Edited Table 1
	Peptide	Sequence a	Length	Structure	Net	%	100%	Reference
			(amino		charge	Hydrophobic	haemolysis of	b
			acids)			residues	RBC
	Dermaseptin	ALWKNMLKGIGKLAGK	16	Helix	+4	50	>150 M	[15]
	S3(1-16)
	Magainin 2	GIGKFLHSAKKFGKAFVGEIMNS 23	Helix	+4	43	>150 g/mL	[10]
	Ranalexin	FLGGLIKIVPAMICAVTKKC	20	Helix with	+3	65	>500 g/mL	[11]
				heptapeptide loop
				from one
				disulphide bridge
	6752	SWFKTKSK	8	Cyclic	+3	25	>400 g/mL	[12]
	(synthetic)
	GS14K4	VKLKVYPLKVKLYP	14	Cyclic	+4	42	200 g/mL	[13]
	Gomesin	ECRRLCYKQRCVTYCRGR	18	-Hairpin with two	+6	33	ca. 20%	[14]
				disulphide bridges			haemolysis
							at 100 M
	RBC, red blood cells.

a

Table 2

 2 Fractional inhibitory concentration indices (FICIs) for combinations of caspofungin or anidulafungin with various antifungal peptides calculated from chequerboard assays of visible growth inhibition of three Candida strains a

	Peptide	Caspofungin

a FICIs were calculated according to Odds

[19]

, where values <0.5 indicate synergy and values 0.5-4 indicate no interaction.

Acknowledgements

The authors are very grateful to Prof. Frank Odds and Dr. Donna MacCallum, Institute of Medical Sciences, University of Aberdeen for performing, and collating/analysing the data from, the mouse model experiments.

Declarations

Funding: PJC and MH were the recipients of a Biotechnology and Biological Sciences Research council studentship (BBS/S/K/2004/11251A). PJC was also partly supported by the NESTech (North East Scotland Technology Seed Fund).

Acknowledgments

The authors are very grateful to Prof. Frank Odds and Dr Donna MacCallum (Institute of Medical Sciences, University of Aberdeen, UK) for performing and collating/analysing the data from the mouse model experiments.

Funding

PJC and MRH were the recipients of a Biotechnology and Biological Sciences Research

Council studentship (BBS/S/K/2004/11251A). PJC was also partly supported by the NESTech (North East Scotland Technology Seed Fund).

Competing interests: None declared

Ethical approval: Not required

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

Edited Figure 5


Adelina Amorim

Luísa Guedes-Vaz

Ricardo Araujo
email: ricjparaujo@yahoo.com

Susceptibility to five antifungals of Aspergillus fumigatus strains isolated from chronically colonised cystic fibrosis patients receiving azole therapy

Keywords:

A c c e p t e d M a n u s c r i p t 3 ABSTRACT Exposure of Aspergillus fumigatus to stressful antifungal therapies may result in decreased susceptibility. The aim of the present work was to evaluate the susceptibility to azole and non-azole antifungals of 159 isolates of A. fumigatus collected from cystic fibrosis (CF) patients receiving azole antifungal therapy. The genetic diversity of the fungal isolates was assessed using microsatellite genotyping, and some strains were found in patient's sputum samples more than 4 years apart. No resistant isolates [minimal inhibitory concentration (MIC)/minimal effective concentration (MEC) ≥ 4 g/mL] were identified to the antifungals amphotericin B, caspofungin, itraconazole and voriconazole. A single A. fumigatus isolate was identified outside of the epidemiological cut-off of 0.25 g/mL for posaconazole. Susceptibility of the recurrent isolates was in agreement with the susceptibility of the first isolate identified (100% essential agreement). Even after azole exposure, several recurrent A. fumigatus strains were detected in the subsequent sputum samples. Development of resistance in A. fumigatus to antifungals appears to be rare among CF patients. However, it remains crucial to evaluate the importance of antifungal agents for allergic fungal diseases.

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

Introduction

Cystic fibrosis (CF) patients are continuously exposed to microorganisms that may colonise their lungs, reducing respiratory function and enhancing pulmonary problems.

Allergic bronchopulmonary aspergillosis (ABPA) is an intense allergic reaction, mainly in response to the presence of Aspergillus fumigatus, which can affect up to 15% of CF patients [START_REF] O'sullivan | Cystic fibrosis[END_REF]. This allergic disease is normally treated with prescription of oral corticosteroid therapy and in a few critical clinical cases azole antifungals may be used [START_REF] O'sullivan | Cystic fibrosis[END_REF][START_REF] Stevens | Allergic bronchopulmonary aspergillosis in cystic fibrosis-state of the art: Cystic Fibrosis Foundation Consensus Conference[END_REF].

Aspergillus fumigatus is one of the most frequently isolated fungi from the lungs of CF patients [START_REF] O'sullivan | Cystic fibrosis[END_REF]. It is commonly susceptible to the commercially available antifungals such as amphotericin B, caspofungin, itraconazole, voriconazole and posaconazole [START_REF] Araujo | Susceptibility of environmental versus clinical strains of pathogenic Aspergillus[END_REF]. It was recently showed that CF patients may be chronically colonised with the same A. fumigatus strain over several years [START_REF] Vanhee | High-resolution genotyping of Aspergillus fumigatus isolates recovered from chronically colonised patients with cystic fibrosis[END_REF][START_REF] De Valk | Molecular typing and colonization patterns of Aspergillus fumigatus in patients with cystic fibrosis[END_REF]. Exposure of A. fumigatus strains to distinct conditions and stressful therapies over long periods may sporadically lead to alterations in their susceptibility and to treatment failure [START_REF] Snelders | Emergence of azole resistance in Aspergillus fumigatus and spread of a single resistance mechanism[END_REF][START_REF] Howard | Frequency and evolution of azole resistance in Aspergillus fumigatus associated with treatment failure[END_REF]. The objective of the present work was to evaluate the susceptibility to azole and non-azole antifungals of 159 isolates of A. fumigatus collected from 11 patients, some of whom had been treated with azole antifungal therapy. 1) received azole antifungal therapy in addition to the recommended antibacterial treatment and oral corticosteroid therapy. Four of the studied patients (patients A, D, E and K shown in Table 1) matched with the modified criteria for diagnosis of ABPA in CF patients [START_REF] Stevens | Allergic bronchopulmonary aspergillosis in cystic fibrosis-state of the art: Cystic Fibrosis Foundation Consensus Conference[END_REF]. performed and antifungal MICs were within the expected limits [START_REF] Barry | Quality control limits for broth microdilution susceptibility tests of ten antifungal agents[END_REF][START_REF] Chaturvedi | Multilaboratory testing of antifungal combinations against a quality control isolate of Candida krusei[END_REF]. Fungal strains were classified accordingly to the recently proposed in vitro breakpoints for amphotericin B, caspofungin, itraconazole and voriconazole (susceptible, MIC < 1 g/mL; intermediate, MIC = 2 g/mL; and resistant, MIC > 4 g/mL) [START_REF] Espinel-Ingroff | Multicenter evaluation of a new disk agar diffusion method for susceptibility testing of filamentous fungi with voriconazole, posaconazole, itraconazole, amphotericin B, and caspofungin[END_REF]. For posaconazole, the fungal strains were classified according to recently proposed epidemiological cut-offs (wild-type, MIC < 0.25 g/mL; non-wild-type, MIC > 0.5 g/mL) [START_REF] Rodriguez-Tudela | Epidemiological cutoffs and cross-resistance to azole drugs in Aspergillus fumigatus[END_REF]. Next, MICs of isolates with the same genotype were compared and were considered in essential agreement when the differences were no more than three dilutions (e.g. 0.5, 1.0 and 2.0 g/mL).

Antifungal therapy

Aspergillus fumigatus genotyping

Microsatellite multiplex polymerase chain reaction (PCR) was performed using previously selected primers allowing the identification of microsatellites based on trinucleotide, tetranucleotide and pentanucleotide motifs located on different chromosomes [START_REF] Araujo | Simple and highly discriminatory microsatellite-based multiplex PCR for Aspergillus fumigatus strain typing[END_REF]. Multiplex PCR was performed using 1 L of genomic DNA (1-5 ng/L), 2.5 L of Multiplex PCR Master Mix (Qiagen, Hilden, Germany) and 0.5 L of a mix with eight primer pairs (final concentration of each primer 2 M) in a final volume of 5 L. The conditions for PCR amplification and sequencing reactions were as previously described [START_REF] Araujo | Simple and highly discriminatory microsatellite-based multiplex PCR for Aspergillus fumigatus strain typing[END_REF].

A c c e p t e d M a n u s c r i p t 9

Statistical analysis

Data were compared at a significance level of 0.05 by analysis of variance (ANOVA) using the Bonferroni correction, and Student's t-test for paired samples. Genotype diversity was calculated by Simpson's index of diversity. Statistical analysis for pair-wise linkage disequilibrium was performed by running the statistical software package Arlequin 3.1 (http://cmpg.unibe.ch/software/arlequin3/).

Results

Among the entire A. fumigatus collection, no resistant isolates (MIC/MEC ≥ 4 g/mL) were identified to the antifungals amphotericin B, caspofungin, itraconazole and voriconazole (Table 1). The susceptibility of a few isolates was classified as intermediate to amphotericin B (3 isolates) and itraconazole (18 isolates), and a single A. fumigatus isolate was identified outside of the epidemiological cut-off of 0.25 g/mL for posaconazole (this last isolate showed a susceptibility of 0.5 g/mL for amphotericin B, 0.06 g/mL for caspofungin, 2 g/mL for itraconazole and 0.5 g/mL for voriconazole). The number of isolates with intermediate susceptibility to itraconazole collected from patients was similar before and after azole treatment (P > 0.05).

The genotype diversity of A. fumigatus found in each patient's sample collection ranged from 0.6 to 1, with an overall diversity of 0.92 when considering the entire group of 159 isolates. There was no significant relationship between A. fumigatus genotype diversity and the patient's diagnosis of ABPA (P > 0.05). Some strains were frequently found in A c c e p t e d M a n u s c r i p t 10 the sputum samples of patients, occasionally more than 4 years apart. A total of 15 strains were repeatedly recovered from the clinical samples (total of 134 isolates) and the susceptibility of the repeated isolates (with the same genotype for all eight microsatellite markers) was in agreement with the susceptibility of the isolate that was first identified (100% essential agreement). Eight of the recurrent (or chronic) strains of A. fumigatus had been exposed to an azole antifungal. Even after azole exposure, some recurrent A. fumigatus (four strains) were still detected in the subsequent sputum samples collected from the patients. Patient B was colonised with three of these recurrent strains and no significant differences were found between the susceptibility of the strains before and after exposure to itraconazole and voriconazole (P > 0.05).

Microvariation events were common among the clinical A. fumigatus isolates collected from six of the patients included in the study; three patients were diagnosed with ABPA. These events were more common between isolates recovered from the same sputum sample (e.g. events 4, 5 or 8), but they could be found in two samples from the same patient collected with more than 3 years apart.

Discussion

Oral corticosteroid therapy remains the first therapy for ABPA and there is still no consensus regarding how, how long or when antifungal drugs should be employed for treatment of patients with ABPA or those colonised with Aspergillus spp. [START_REF] Stevens | Allergic bronchopulmonary aspergillosis in cystic fibrosis-state of the art: Cystic Fibrosis Foundation Consensus Conference[END_REF][START_REF] Schoeni | To treat or not to treat with antifungal agents?[END_REF]. Patients included in this study received azole antifungal therapy due to serious haemoptysis A c c e p t e d M a n u s c r i p t 11 and/or decrease of respiratory function and clinical deterioration following administration of antibacterial treatment and/or corticosteroid therapy. In all cases there was verified clinical progress and favourable outcome. Antifungals have been seldom used for therapy among CF patients, but the long-standing and significant adverse effects of corticosteroid therapy, the difficulties associated with a firm diagnosis of ABPA and the clinical deterioration of chronically ill patients may result in the near future in an increase of antifungal usage among CF patients [START_REF] Schoeni | To treat or not to treat with antifungal agents?[END_REF].

During the last decade there has been an increase, although infrequent, in the description of itraconazole resistance among A. fumigatus isolates obtained from patients with invasive fungal infections receiving antifungal drugs [START_REF] Snelders | Emergence of azole resistance in Aspergillus fumigatus and spread of a single resistance mechanism[END_REF][START_REF] Rodriguez-Tudela | Epidemiological cutoffs and cross-resistance to azole drugs in Aspergillus fumigatus[END_REF]. Resistance to voriconazole or posaconazole has been more rarely reported. Recently, the association of A. fumigatus azole resistance in allergic diseases was described resulting from prior exposure to an antifungal azole agent [START_REF] Howard | Frequency and evolution of azole resistance in Aspergillus fumigatus associated with treatment failure[END_REF]. In all cases, treatment failure was reported and several A. fumigatus strains were identified as resistant to itraconazole, voriconazole and/or posaconazole. Conversely, in this study we could not detect any resistant A. fumigatus isolate to the five tested antifungals, azoles or non-azoles (a few fungal isolates were intermediate to amphotericin B or itraconazole). Nevertheless, we did not find unfavourable outcomes among the 11 evaluated patients.

Microsatellite genotyping results demonstrated that cystic CF fibrosis patients may be chronically colonised with the same strains, sometimes over several years as previously described [START_REF] Vanhee | High-resolution genotyping of Aspergillus fumigatus isolates recovered from chronically colonised patients with cystic fibrosis[END_REF][START_REF] De Valk | Molecular typing and colonization patterns of Aspergillus fumigatus in patients with cystic fibrosis[END_REF]. In any case, exposure to an antifungal azole did not reduce the However, we can question whether the use of azole antifungal therapy was really effective. One-half of the recurrent A. fumigatus strains were detected in the subsequent sputum samples collected from patients. The limited number of studied patients and, in particular, the fact that all patients showed a good response to the therapy may limit additional conclusions. Nevertheless, we can assume with the present results that azole antifungal resistance is not a common event among A. fumigatus isolates collected from CF patients. Aspergillus fumigatus isolates may become resistant to antifungals only under particular conditions, uncommon therapeutic options and/or unusual structures.

An example is mould biofilm that may be present in the CF lung and has already been described as exhibiting decreased antifungal drug susceptibility and longer viability compared with planktonic and sessile forms [START_REF] Seidler | Aspergillus fumigatus forms biofilms with reduced antifungal drug susceptibility on bronchial epithelial cells[END_REF].

At present, it is crucial to re-evaluate several clinical therapies in order to avoid the appearance and dissemination of resistant strains. Azole antifungals remain the single oral solution available for aspergillosis and the development of resistance in these fungi seems to be rare. None the less, several strains can persist in the lungs of CF patients even after administration of azole antifungal therapy, questioning the significance of such treatment. It remains essential to define the clinical situations that indeed require this therapeutic alternative. Additionally, we also need to evaluate the importance of antifungal agents for allergic diseases in situations of failure of bacterial and corticosteroid treatments.

2).

Edited Table 1

  A group of 11 patients admitted to Hospital S. João (Porto, Portugal) and diagnosed with CF were studied. The diagnosis of CF was confirmed by screening CFTR mutations and positive evaluation of the sweat test. Patient age ranged from 15 years to 51 years. Patients were selected for this study following confirmation of chronic colonisation with A. fumigatus (more than three sputum samples with A. fumigatus in a previous year). Sputum samples were then collected from the patients between January 2005 and May 2009 (with a minimum of two samples from each patient at different times). Seven patients never received antifungal treatment, whilst four of the patients (patients B, C, D and E shown in Table

  Patient B received itraconazole between December 2007 and April 2008 (5 months) at a daily dose of 200 mg due to a serious decrease of respiratory function and chronic colonisation with A. fumigatus and Scedosporium apiospermum. In April 2008, an infection by tuberculosis mycobacteria was diagnosed and treated and itraconazole therapy was suspended to avoid liver dysfunction. Because the decrease in respiratory

A

  c c e p t e d M a n u s c r i p t 6 function persisted, patient B started voriconazole therapy in June 2009 (200 mg/12 h). The patient is currently stabilised and remains on voriconazole therapy. Patient C received itraconazole between November 2006 and September 2007 (12 months) at a daily dose of 400 mg due to a decrease of respiratory function and clinical and radiological deterioration (mycetoma). The patient was chronically colonised with A. fumigatus and S. apiospermum. The clinical picture persisted after 1 year of itraconazole and in September 2007 the patient started voriconazole at 200 mg/12 h. Sputum samples showed the absence of fungal colonisation following voriconazole treatment. Between January and March 2008 the patient developed hepatitis and voriconazole therapy was suspended. Patient C re-started voriconazole therapy in April 2008 without any further complication and remains on the antifungal treatment until now (August 2009). Patient D received voriconazole therapy for 12 months (from September 2007 to September 2008) at a dose of 200 mg/12 h due to serious haemoptysis and diagnosis of ABPA. The patient recovered and is currently stabilised. Patient E received voriconazole therapy for 8 months (between June 2006 and February 2007; dose of 200 mg/12 h) due to ABPA, with a favourable outcome. Presently (August 2009), the patient is receiving itraconazole therapy (daily dose of 200 mg initiated in September 2008) due to a serious haemoptysis and no criteria for ABPA.The patient is presently stabilised.

  A c c e p t e d M a n u s c r i p t 72.3. Fungal organismsSputum samples were cultured in Sabouraud agar for 10 days at 30 C and 37 C. A total of 159 isolates of A. fumigatus were recovered from the clinical samples. The isolates were identified based upon macroscopic and microscopic morphological characteristics following standard mycological procedures. Two A. fumigatus reference strains (ATCC 46645 and MYA 772) were included in the study. Prior to DNA isolation, moulds were grown for 5 days on Sabouraud dextrose agar slants (Difco, Detroit, MI) at 30 C. A sodium hydroxide-based method was used to extract DNA from conidia, as previously described[START_REF] Araujo | Simple and highly discriminatory microsatellite-based multiplex PCR for Aspergillus fumigatus strain typing[END_REF]. DNA was suspended in 50 L of sterile water and stored at -20 C.2.4. Antifungal susceptibility testingDetermination of minimal inhibitory concentrations (MICs) of amphotericin B (Bristol-Myers SP, Dublin, Ireland), caspofungin (Merck Sharp & Dohme, Whitehouse Station, NJ), itraconazole (Janssen-Cilag, High Wycombe, UK), voriconazole (Pfizer Inc., New York, NY) and posaconazole (Schering-Plough Farma, Cacém, Portugal) was performed according to the Clinical and Laboratory Standards Institute broth microdilution method[START_REF]method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard[END_REF]. MICs were defined as the absence of visible growth after 48 h in the presence of the antifungals amphotericin B, itraconazole, voriconazole and posaconazole. For caspofungin, the minimal effective concentration (MEC) was

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

 8 as previously recommended[START_REF] Araujo | Susceptibility of environmental versus clinical strains of pathogenic Aspergillus[END_REF]. Quality control strains Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were included each time testing was

A

  c c e p t e d M a n u s c r i p t 12 susceptibility of A. fumigatus genotypes chronically recovered from patients and, occasionally, among isolates that showed microvariation (disparity in a single marker).

Fa

  Susceptibility to amphotericin B (AMB), caspofungin (CAS), itraconazole (ITC), voriconazole (VRC) and posaconazole (PSC) of 159 Aspergillus fumigatus isolates collected from the sputa of 11 cystic fibrosis patients Patient -0.125 0.5-2 0.25-0.5 0.125 E (13) b 0.5-1 ≤0.03-0.06 0.5-1 0.25-0.5 0.125-0.25 Number of isolates recovered from each patient in parenthesis. b Patients B, C, D and E received antifungal azole therapy (details in Section 2.

Acknowledgments

The authors are grateful to Maria Luz Dias and Isabel Santos for excellent technical assistance.

Funding

This work was supported by grants from Fundação Calouste Gulbenkian (no. 35-9924-S/2009), University of Porto and Santander Totta. RA was co-financed by the European Social Fund. IPATIMUP is partially supported by Fundação para a Ciência e Tecnologia (FCT), Programa Operacional Ciência e Inovação (POCI) and Programa Operacional do Potencial Humano (POPH -QREN).

Competing interests

None declared.

Ethical approval

Not required.


Danuta Dzierżanowska

Wanda Kamińska

Katarzyna Semczuk

Dariusz Borowiec

Michał Matysiak

Anna Szumała-Kąkol

Rafał Gierczyński

Jan A Patzer

Carriage of genes for various extended-spectrum -lactamases: a novel resistance strategy of Klebsiella pneumoniae in Poland

Keywords:

E-mail address: j.patzer@czd.pl (J.A. Patzer).

A c c e p t e d M a n u s c r i p t ABSTRACT Among 110 randomly sampled strains from a collection of 247 extended-spectrum lactamase (ESBL)-producing clinical isolates of Klebsiella pneumoniae collected from hospitalised children in three paediatric hospitals in Poland, 64 strains (58.2%) with multiple ESBLs were found, including five non-clonal strains (4.5%) harbouring bla genes for ESBLs of three families (CTX-M, SHV and TEM). This is the first report of the emergence of triple ESBL-producing K. pneumoniae in Poland. In addition, K. pneumoniae strains harbouring bla genes for TEM-130 and TEM-132 ESBLs were detected in Poland for the first time. Epidemiological analysis of the multiple ESBLproducing K. pneumoniae isolates by pulsed-field gel electrophoresis (PFGE) revealed a relatively high genetic diversity between isolates producing the same combination of enzymes. Clonally related strains were uncommon.

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

Introduction

Extended-spectrum -lactamases (ESBLs) are the predominant resistance agents to oxyimino--lactams in clinical isolates of Gram-negative bacteria. In general, ESBLs are capable of hydrolysing penicillins, broad-spectrum cephalosporins and aztreonam. Owing to their rapid dissemination and diversity, ESBLs are an increasing problem worldwide [START_REF] Bradford | Extended-spectrum -lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat[END_REF]. Genes encoding ESBLs usually map on mobile genetic elements within large plasmids, which often spread horizontally. Initially, resistance to oxyimino-cephalosporins in Gram-negative rods was mainly due to dissemination of TEM-and SHV-type ESBLs. During the last 10 years, CTX-M-type ESBLs have become predominant [START_REF] Livermore | CTX-M: changing the face of ESBLs in Europe[END_REF]. ESBLs are frequently reported to be coproduced with penicillinases in clinical isolates of Enterobacteriaceae. Klebsiella pneumoniae strains producing multiple ESBLs were isolated for the first time in Chicago in 1990 and 1991. The strains were found to produce two different ESBLs of the same family (TEM-12 and TEM-10) in a single isolate [START_REF] Bradford | Multiply resistant Klebsiella pneumoniae strains from two Chicago hospitals: identification of the extended-spectrum TEM-12 and TEM-10 ceftazidime-hydrolyzing -lactamases in a single isolate[END_REF]. Later, K. pneumoniae isolates producing multiple ESBLs of different families (TEM and SHV) were collected in Durban, South Africa [START_REF] Essack | Complexity and diversity of Klebsiella pneumoniae strains with extended-spectrum -lactamases isolated in 1994 and 1996 at a teaching hospital in Durban, South Africa[END_REF]. Strains harbouring bla genes encoding for ESBLs of two or three different families emerged soon after [START_REF] Paterson | Extended-spectrum -lactamases in Klebsiella pneumoniae bloodstream isolates from seven countries: dominance and widespread prevalence of SHV-and CTX-M-type -lactamases[END_REF]. In Poland, K. pneumoniae isolates simultaneously expressing CTX-M-3 and SHV-or TEM-type ESBLs were reported in 2002 [START_REF] Baraniak | Countrywide spread of CTX-M-3 extended-spectrum -lactamase-producing microorganisms of the family Enterobacteriaceae in Poland[END_REF]. The aim of this study was to determine the prevalence of multiple ESBL-producing K. pneumoniae isolates in three selected paediatric hospitals in Poland. Here we report 64 K. pneumoniae clinical isolates carrying bla genes for simultaneous expression of two or three different ESBLs.

Polymerase chain reaction (PCR) screening for extended-spectrum -lactamse genes and DNA sequencing

The conserved core regions of the bla CTX-M , bla SHV and bla TEM genes, which represent the most commonly reported ESBL families in Poland, were amplified by PCR [START_REF] Jarlier | Extended broad-spectrum lactamases conferring transferable resistance to newer -lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns[END_REF] to identify isolates genetically competent to produce -lactamases of different families simultaneously. Further, the exact ESBL genotypes were determined by DNA sequencing. Briefly, the core regions of the bla genes and the overlapping upstream and downstream gene fragments were amplified by PCR using the primers listed in Table 1. Both DNA strands of the PCR amplicons were and CLC Free Workbench V 6.0 software were used for processing of the sequencing data and to assemble the complete gene sequences.

Antimicrobial susceptibility testing

Minimum inhibitory concentrations of cefotaxime, ceftazidime, cefepime, piperacillin/tazobactam, ciprofloxacin, imipenem, meropenem and ertapenem were determined for each multiple ESBL-producing isolate by the agar dilution method on Iso-Sensitest agar [START_REF] Gierczyński | The molecular characterisation of the extended-spectrum -lactamase (ESBL) producing strain of Salmonella enterica serovar Mbandaka isolated in Poland[END_REF]. Quality control was performed using strains Escherichia coli ATCC 25922 and K. pneumoniae ATCC 70060.

Pulsed-field gel electrophoresis (PFGE)

Isolates revealing a multiple ESBL genotype were subjected to PFGE typing with a CHEF DR II System (Bio-Rad, Hercules, CA). Briefly, agarose-embedded genomic DNA was digested with XbaI (Fermentas, Vilnius, Lithuania) and digestion products were separated in 1% w/v agarose gel (Pulsed Field Certified Agarose; Bio-Rad) in TBE buffer [45 mM Tris-base, 45 mM boric acid, 1 mM ethylene diamine tetra-acetic acid (EDTA), pH 8.0]. Electrophoresis was carried out at 6 V/cm at 14 C for 10 h with a switch time of 5 s to 15 s, followed by 10 h with a switch time of 15 s to 45 s using a CHEF DR II System.

A dendrogram illustrating the genetic similarity of the multiple ESBL-producing isolates was generated using the Molecular Analyst Program (Bio-Rad).

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

Results and discussion

Among the 110 tested strains of K. pneumoniae, 46 carried a single bla gene encoding for ESBL, whilst 64 strains harboured bla genes for at least two ESBLs of different families (Table 2). The majority of isolates producing a single ESBL harboured the bla CTX-M-3 gene (39/46) and the remaining isolates carried the following genes: bla SHV-5 (n = 3); bla CTX-M-15 (n = 2); bla TEM-47 (n = 1); and bla TEM-48 (n = 1). Fifty-nine isolates and five isolates, respectively, were found to carry two or three bla genes for different ESBL families.

The combination of bla CTX-M-3 with bla TEM-48 or bla SHV-5 predominated in isolates producing two ESBLs (dual ESBLs). The less common bla CTX-M-15 gene was found in combination with bla TEM-48 or bla TEM-47 , found in strains isolated from CMHI only. The bla TEM genes for TEM-130 and TEM-132 were detected in K. pneumoniae strains producing two ESBLs. Interestingly, these two TEM variants have not been reported in Poland. TEM-130 has not yet been fully described. Its amino acid sequence differs from TEM-129 by a single substitution (D35P) (http://www.lahey.org/Studies/temtable.asp). Therefore, in this study TEM-130 was considered as the ESBL. TEM-132, first reported in 2005, was originally found in neighbouring Slovakia [START_REF]Performance standards for antimicrobial susceptibility testing[END_REF]. Notably, 36 of the dual ESBL-producing K. pneumoniae isolates tested in this study were collected in CMHI, whereas only 23 were isolated from patients in the two regional hospitals PMUH and WMUH. All the isolates genetically competent to produce three ESBLs (triple ESBL genotype) were collected exclusively from CMHI patients. They had the following four genotypes: (i) bla CTX-M-3 , bla TEM-47 and bla SHV-5 ; (ii) bla CTX-M-3 , bla TEM-48 and bla SHV-5 ; (iii) bla CTX-M-3 , bla TEM-86 and bla SHV-5 ; and (iv) bla CTX-M-15 , bla TEM-48 and bla SHV-5 . The prevalence of isolates

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

showing a multiple ESBL genotype in CMHI was higher (67.2%) than in PMUH (57.9%) and WMUH (40.0%). In contrast to the two regional hospitals, CMHI is the reference paediatric hospital to which serious cases from the whole of Poland are admitted. Before admission to CMHI, many patients had prolonged or multiple hospitalisation events in a primary-care hospital, often connected with one or two antibiotic therapies.

All the multiple ESBL-producing K. pneumoniae strains were susceptible to imipenem, meropenem and ertapenem, and some were susceptible to piperacillin/tazobactam (Table 2). These strains were also susceptible to ciprofloxacin, which is in contrast to reports by Paterson et al. [START_REF] Zarnayová | Survey of Enterobacteriaceae producing extended-spectrum -lactamases in a Slovak hospital: dominance of SHV-2a and characterization of TEM-132[END_REF] and Damjanova et al. [START_REF] Paterson | Epidemiology of ciprofloxacin resistance and its relationship to extended-spectrum -lactamase production in Klebsiella pneumoniae isolates causing bacteremia[END_REF] who found a close relationship between ESBL production and ciprofloxacin resistance in K. pneumoniae. This may be due to the strongly limited consumption of fluoroquinolones in paediatric hospitals.

It is noteworthy that the majority of the multiple ESBL genotypes observed in this study were composed of genes encoding for ESBL variants that are common in Poland [START_REF] Damjanova | Expansion and countrywide dissemination of ST11, ST15 and ST147 M a n u s c r i p t ciprofloxacin-resistant CTX-M-15-type -lactamase-producing Klebsiella pneumoniae epidemic clones in Hungary in 2005-the new 'MRSAs'?[END_REF][START_REF] Baraniak | Evolution of the TEM-type extended-spectrum -lactamases in clinical Enterobacteriaceae strains in Poland[END_REF]. This finding may argue for self-dependent acquisition of the most common genes encoding ESBL by K. pneumoniae clinical isolates. It was particularly interesting to determine whether K. pneumoniae isolates sharing the same multiple ESBL genotype are clonal. Therefore, all 110 isolates were subjected to PFGE. The majority of the multiple ESBL-producing isolates from CMHI were non-clonal with a genetic similarity <70% (Fig. 1). In contrast, the multiple ESBL-producing isolates from both local hospitals (PMUH and WMUH) were mostly clonal. Two clones covering six and three isolates and only two non-clonal ones were found in PMUH.

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

Similarly, five clones of two isolates each, together with only two non-clonal isolates, were detected among the multiple ESBL-producing isolates from WMUH. Findings from these two local hospitals may argue for an enhanced epidemic potential of K. pneumoniae harbouring multiple bla genes for ESBLs.

Multiple ESBL-producing K. pneumoniae appear to occur worldwide. Among bloodstream K. pneumoniae isolates collected in 12 hospitals in Argentina, Australia, Belgium, South Africa, Taiwan, Turkey and the USA, up to 19.2% were found to produce multiple ESBLs [START_REF] Paterson | Extended-spectrum -lactamases in Klebsiella pneumoniae bloodstream isolates from seven countries: dominance and widespread prevalence of SHV-and CTX-M-type -lactamases[END_REF]. Isolates of such a phenotype may be even more common. Recently, it has been reported that 44% of ESBL-producing isolates of E. coli, K. pneumoniae and Proteus mirabilis harboured bla genes for multiple ESBLs [START_REF] Jones | Characterization and sequence analysis of extended-spectrum--lactamase-encoding genes from Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis isolates collected during tigecycline phase 3 clinical trials[END_REF].

In Poland, the last country-wide survey on ESBL-producing Enterobacteriaceae was conducted between November 2003 and January 2004 [START_REF] Damjanova | Expansion and countrywide dissemination of ST11, ST15 and ST147 M a n u s c r i p t ciprofloxacin-resistant CTX-M-15-type -lactamase-producing Klebsiella pneumoniae epidemic clones in Hungary in 2005-the new 'MRSAs'?[END_REF]. This survey showed that 1.5% of 264 isolates collected in regional secondary-care hospitals co-produced two different ESBLs (CTX-M-15 and SHV-5). No isolates capable of producing three different ESBLs were found. In contrast, the results of this study showed that 58.2% of ESBL-producing K. pneumoniae isolates revealed a multiple ESBLs genotype. This could be explained by differences in studied patients, bacterial species and the study period. The current study was focused on K. pneumoniae, which is known to develop novel resistance traits rapidly. The tested isolates were collected from patients hospitalised between 2000 and 2007, mostly from the reference hospital (CMHI). It is noteworthy that the majority of the multiple ESBL-producing isolates (82.8%) were collected after 2003. All the triple ESBL-producing isolates emerged in

  author. Tel.: +48 22 815 7271; fax: +48 22 815 7275.

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

 2 Materials and methods2.1. Bacterial strainsA total of 110 strains was randomly selected from a collection of 247 ESBLproducing K. pneumoniae isolates recovered from various clinical specimens from children hospitalised during 2000-2007 in the reference hospital Children's Memorial Health Institute (CMHI) in Warsaw (n = 137), the neonatology ward in Poznań Medical University Hospital (PMUH) (n = 61) and the Clinic of Paediatrics, Haematology and Oncology in the Warsaw Medical University Hospital (WMUH) (n = 49). All selected strains were confirmed as ESBL-producers by the double-disk synergy test using a combination of cefotaxime, ceftazidime and amoxicillin/clavulanic acid [7].

  sequenced using an automated fluorescent DNA sequencer ABI PRISM 310 (Applied Biosystems, Foster City, CA) and a BigDye ® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) in accordance with the manufacturer's A c c e p t e d M a n u s c r i p t instructions. Primers for PCR and DNA sequencing were the same. FinchTV V 1.4

A

  c c e p t e d M a n u s c r i p t CMHI after 2004. In light of this, it may be supposed that the accumulation of multiple bla genes for different ESBLs in K. pneumoniae is tending to increase in Poland. Further studies are therefore required to deepen our knowledge of mechanisms leading to the selection and dissemination of multiple ESBL-expressing isolates. A c c e p t e d M a n u s c r i p t

Fig. 1 .

 1 Fig. 1. Dendrogram of the clonal relationship of multiple extended-spectrum -

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

 2 Molecular characterisation of the bla genes among multiple extended spectrum -lactamse (ESBL)-producing Klebsiella pneumoniae (n = 64) and activity of selected antibiotics against tested isolates

Table 1

 1 Nucleotide sequences of polymerase chain reaction (PCR) primers used in the study

	Gene	Fragment	Forward (5'3')	Reverse (5'3')	Size (bp) Reference
	bla CTX-M Upstream	CTTTCGGAAGCATAAAATCGG	CTCAGCCAGTGACATCGTCC	472	This study
	bla CTX-M Core	TGCAGTACCAGTAAGGTGATGG CCAGATAACCGCGATATCGTTG 546	[8]
	bla CTX-M Downstream TGGTGACATGGATGAAAGGC	GCACTTTTGCCGTCTAAGGC	311	This study
	bla TEM	Upstream	CGCTCATGAGACAATAACC	AAGGATCTTCACCTAGATCC	379	This study
	bla TEM	Core	AGTTCTGCTATGTGGTGCGG	GCTAGAGTAAGTAGTTCGCC	387	[8]
	bla TEM	Downstream AGCCATACCAAACGACGAGC	AAGGATCTTCACCTAGATCC	439	This study
	bla SHV	Upstream	AAGAGGAATTGTGAATCAGC	GATCCTGCTGGCGATAGTGG	387	This study
	bla SHV	Core	GAGCGAAAGATCCACTATCG	CCTCATTCAGTTCCGTTTCC	241	[8]
	bla SHV	Downstream GATCGGCGACAACGTCACC	TTAGCGTTGCCAGTGCTCGATC 412	This study

Funding

This work was supported by the Polish State Committee for Scientific Research (grant number 2 P05D 057 29).

Competing interests

None declared.

Ethical approval

Not required.