Robert-Jan Hassing

Godfred A Menezes

Wilfred Van Pelt

Pieter L Petit

Perry J Van Genderen

Wil H F Goessens

Analysis of mechanisms involved in reduced susceptibility to ciprofloxacin in Salmonella enterica serotypes Typhi and Paratyphi A isolates from travellers to Southeast Asia

E-mail address: p.van.genderen@havenziekenhuis.nl (P.J. van Genderen).

A c c e p t e d M a n u s c r i p t 3 ABSTRACT Owing to multidrug resistance, quinolones and third-generation cephalosporins are currently used as key antibiotics to combat Salmonella organisms. Therapy failure due to reduced ciprofloxacin susceptibility has been reported in endemic areas, but also in imported disease. Different bacterial resistance mechanisms may result in reduced ciprofloxacin susceptibility. In this study, the presence and expression of different resistance mechanisms resulting in reduced minimum inhibitory concentrations (MICs) for ciprofloxacin were evaluated in 23 blood culture-derived Salmonella enterica serotypes Typhi and Paratyphi A organisms from ill-returned travellers to Asia. The presence of mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene as well as an activated efflux pump and plasmidmediated quinolone resistance genes was determined. Resistance selection during therapy and the clonal relatedness of all isolates were established. Efflux pump inhibition did not appear to affect the MICs of ciprofloxacin and activity of the efflux pump appeared to be specific for nalidixic acid. Repeated exposure of the isolates to ciprofloxacin did not result in a significant increase in the MICs for ciprofloxacin.

Repetitive sequence-based polymerase chain reaction (rep-PCR) profiles identified five different genotypes, but no correlation with resistance was observed. However, a significant relation was found with geographic region; reduced ciprofloxacin susceptibility was only found in travellers returning from India and Pakistan. All isolates with reduced ciprofloxacin susceptibility had a mutation at position 83 in the QRDR region of the gyrA gene. Plasmid-mediated quinolone resistance was not found. These findings confirm that the reduced ciprofloxacin MIC in S. Typhi and S.

Paratyphi A is solely due to an amino acid substitution in the QRDR 'cluster' of the gyrA gene.

Introduction

Resistance of Salmonella spp. to ampicillin, chloramphenicol and trimethoprim/sulfamethoxazole has been described during the last 20 years. Owing to this multidrug resistance, quinolones and third-generation cephalosporins are currently in use as key antibiotics to combat these multiresistant organisms [START_REF] Bhan | Typhoid and paratyphoid fever[END_REF]. As a consequence of the use of quinolones and cephalosporins, there have been reports describing clinical treatment failure following administration of ciprofloxacin to patients with typhoid fever owing to reduced ciprofloxacin susceptibility [defined as a minimum inhibitory concentration (MIC) of 0.125-1 g/mL]. This reduced susceptibility is found not only in endemic areas but also in developed countries such as the USA and UK, reflecting a global problem [START_REF] Wain | Quinoloneresistant Salmonella typhi in Viet Nam: molecular basis of resistance and clinical response to treatment[END_REF][START_REF] Threlfall | Decreased susceptibility to ciprofloxacin in Salmonella enterica serotype Typhi, United Kingdom[END_REF].

Bacterial resistance to quinolones in Salmonella spp. may result from different resistance mechanisms. Quinolone resistance is usually associated with mutations in the target site (DNA gyrase), most commonly in the quinolone resistance-determining region (QRDR) of the GyrA subunit, conferring resistance to single-step mutants [START_REF] Piddock | Mechanisms of resistance to fluoroquinolones: state-of-the-art 1992-1994[END_REF].

On the other hand, reduced susceptibility to quinolones is mostly due to mechanisms such as active efflux via efflux pumps as well as alterations in the expression of outer membrane proteins [START_REF] Cebrián | Characterization of Salmonella spp. mutants with reduced fluoroquinolone susceptibility: importance of efflux pump mechanisms[END_REF]. Plasmid-mediated quinolone resistance has also emerged in non-typhoidal Salmonella enterica strains resulting in reduced ciprofloxacin susceptibility; this mechanism has not yet been observed in typhoidal Salmonella spp. Outer membrane protein-deficient Salmonella spp. have only been described incidentally, resulting in increased MICs (>200 g/mL) for chloramphenicol [START_REF] Toro | Clinical isolate of a porinless Salmonella typhi resistant to high levels of chloramphenicol[END_REF].

Materials and methods

Bacterial isolates

Antimicrobial susceptibility

Susceptibility of the isolates was determined using the VITEK-2 system (bioMérieux sa, Marcy l'Etoile, France). MICs for nalidixic acid and ciprofloxacin were determined

Polymerase chain reaction (PCR) screening and sequence analysis

The QRDR of gyrA was amplified by PCR using the primers described by Kariuki et al. [START_REF] Kariuki | Characterization of multidrug-resistant typhoid outbreaks in Kenya[END_REF]. Products resulting from amplifications were subjected to sequencing using a 3100 ABI Prism Genetic Analyzer (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands). Analysis and comparison of nucleotide and amino acid sequence data were carried out using MegAlign software (DNASTAR Inc., Madison, WI) and programs available from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). Presence of the qnrA, qnrB, qnrS and aac(6')-Ib-cr genes was determined by PCR [START_REF] Wu | Prevalence of plasmid-mediated quinolone resistance determinants QnrA, QnrB, and QnrS among clinical isolates of Enterobacter cloacae in a Taiwanese hospital[END_REF][START_REF] Robicsek | Fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase[END_REF]. The presence of integrons was determined using the method described by Lévesque et al. [START_REF] Lévesque | PCR mapping of integrons reveals several novel combinations of resistance genes[END_REF].

Molecular typing

Clonal relatedness of all the isolates was established using the repetitive sequencebased PCR (rep-PCR) DiversiLab ® Microbial Typing System (bioMérieux) according

Results and discussion

During the last decade, treatment failures with ciprofloxacin have been increasingly reported. These failures have been associated with infection with S. Typhi and S.

Paratyphi A strains that are resistant to nalidixic acid with decreased susceptibility to ciprofloxacin [START_REF] Wain | Quinoloneresistant Salmonella typhi in Viet Nam: molecular basis of resistance and clinical response to treatment[END_REF][START_REF] Threlfall | Decreased susceptibility to ciprofloxacin in Salmonella enterica serotype Typhi, United Kingdom[END_REF][START_REF] Slinger | Suboptimal clinical response to ciprofloxacin in patients with enteric fever due to Salmonella spp. with reduced fluoroquinolone susceptibility: a case series[END_REF]. Studies describing mechanisms of fluoroquinolone resistance demonstrate only one or two specific mechanisms. However, several mechanisms of resistance can result in reduced susceptibility to ciprofloxacin [START_REF] Piddock | Mechanisms of resistance to fluoroquinolones: state-of-the-art 1992-1994[END_REF]. Moreover, a combination of resistance mechanisms could also lead to enhancement of each of the separate mechanisms resulting in increased MICs. As mentioned previously, studies describing a systematic analysis of fluoroquinolone resistance mechanisms in different S. Typhi and S. Paratyphi A isolates are rare [START_REF] Gaind | Molecular characterization of ciprofloxacin-resistant Salmonella enteric serovar Typhi and Paratyphi A causing enteric fever in India[END_REF]. Therefore, we have systematically evaluated the presence and expression of different mechanisms of resistance resulting in reduced MICs for ciprofloxacin in 23 Salmonella clinical isolates.

Of the 23 isolates, 14 (60.9%) were resistant to nalidixic acid (NAL R ) and displayed reduced MICs for ciprofloxacin ranging from 0.25 g/mL to 2 g/mL, except for one isolate (6838) that had a ciprofloxacin MIC of 32 g/mL. The remaining nine isolates (39.1%) were all nalidixic acid-susceptible (NAL S ) and had low MICs for ciprofloxacin (0.03 g/mL) (Fig. 1). One of the mechanisms resulting in reduced susceptibility to quinolones may be the presence of activated efflux pumps. To detect the presence of A c c e p t e d M a n u s c r i p t 9 these pumps, a phenotypic method was applied by determining MICs for nalidixic acid and ciprofloxacin in the presence and absence of the EPI PAN. In most isolates, moderate efflux activity was determined for nalidixic acid resulting in MIC reductions of 3-4 steps in the presence of the EPI. However, the activity of this particular efflux pump(s) does not appear to affect the MICs of ciprofloxacin, indicating that the activity of this pump is specific, at least for nalidixic acid. As this phenomenon is not exclusively found in NAL R isolates, it is concluded that this specific efflux pump(s) is not causing the increased MICs for ciprofloxacin in the NAL R isolates (data not shown). Patients infected with isolates with reduced susceptibility to fluoroquinolones react less favourably on the initiated therapy [START_REF] Slinger | Suboptimal clinical response to ciprofloxacin in patients with enteric fever due to Salmonella spp. with reduced fluoroquinolone susceptibility: a case series[END_REF]. This might be due to the fact that the strains that are already resistant to nalidixic acid may require fewer exposures to fluoroquinolones to develop high-level resistance to ciprofloxacin than strains that are fully ciprofloxacin-susceptible [START_REF] Cebrián | Characterization of Salmonella spp. mutants with reduced fluoroquinolone susceptibility: importance of efflux pump mechanisms[END_REF].

To check for resistance selection during therapy, a selected number of isolates were exposed for two rounds to increasing concentrations of ciprofloxacin. However, this limited exposure did not result in a significant increase in the MICs of ciprofloxacin. Also, efflux pump activity did not change after these two rounds of selection/induction by repeated exposure to ciprofloxacin (data not shown). In fact, the same base level efflux pump activity was observed for chloramphenicol (efflux indicator antibiotic) as was already demonstrated for nalidixic acid. Thus, the impaired clinical response is not due to resistance selection during antibiotic treatment but may be due to unfavourable pharmacodynamic parameters resulting in impaired killing. Isolate 6838, with a ciprofloxacin MIC of 32 g/mL, for had a double mutation. In addition to the mutation at position 83, a substitution of asparagine to glycine at position 87 was also demonstrated. Mutations in gyrA outside of the QRDR were observed both in NAL R and NAL S isolates (data not shown). These data are in full agreement with the data of Nair et al. [START_REF] Nair | Molecular analysis of fluoroquinolone-resistant Salmonella Paratyphi A isolate, India[END_REF] who demonstrated that a single gyrA mutation at Ser-83 alone resulted in resistance to nalidixic acid and reduced susceptibility to ciprofloxacin (MICs of 0.125-0.25 g/mL). Therefore, it was concluded that Ser-83 is an important site for determining fluoroquinolone resistance within S. Typhi and S. Paratyphi A isolates. Complete resistance to ciprofloxacin (MIC > 4 g/mL) has been reported by Gaind et al. [START_REF] Gaind | Molecular characterization of ciprofloxacin-resistant Salmonella enteric serovar Typhi and Paratyphi A causing enteric fever in India[END_REF] and is due to a double mutation in the QRDR region.

In this investigation, the presence of plasmid-encoded qnr genes resulting in reduced susceptibility to quinolones in Salmonella spp. was also studied [START_REF] Gay | Plasmid-mediated quinolone resistance in non-Typhi serotypes of Salmonella enterica[END_REF]. Expression of these genes is phenotypically hard to detect as they have a moderate effect on ciprofloxacin MICs even in combination with the aac(6')-Ib-cr gene. In the 23 isolates To study the genetic relationship between the NAL R and NAL S clusters, all isolates were analysed using a commercial rep-PCR system. Results of rep-PCR (Fig. 1) showed three different clusters of 8, 2 and 11 isolates as well as 2 unique isolates. In clusters A and D consisting of 8 and 11 isolates, respectively, susceptibility to the quinolones was randomly distributed, thereby demonstrating that resistance to nalidixic acid, i.e. reduced ciprofloxacin susceptibility, is not linked to a distinct cluster. Isolates belonging to cluster A were all S. Typhi isolates, whereas cluster D comprised S. Paratyphi A isolates. The only consistent correlation found related to the country of acquisition of the infection, i.e. resistance to nalidixic acid and reduced susceptibility to ciprofloxacin was only found in travellers to India and Pakistan, but not in travellers to Indonesia and Cambodia. However, these geographical differences are not always that clear, as NAL R isolates have also been reported in Vietnam [START_REF] Wain | Quinoloneresistant Salmonella typhi in Viet Nam: molecular basis of resistance and clinical response to treatment[END_REF]. However, based on these data it seems at least indicated to treat

  author. Tel.: +31 10 404 3305; fax: +31 10 412 1645.

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

 6 to increased travel to endemic areas as well as migrant workers originating from endemic countries, The Netherlands is also confronted with patients not responding to empirical therapy with ciprofloxacin. In this retrospective study, S. enterica serotypes Typhi and Paratyphi A isolates were systematically characterised with respect to the presence and expression of different mechanisms of resistance causing reduced susceptibility to fluoroquinolones in 23 consecutive patients (travellers and migrant workers) diagnosed at the Institute for Tropical Diseases of Harbour Hospital-Rotterdam (The Netherlands) with blood culture-derived S. Typhi or S. Paratyphi A infection.

  In total, 23 Salmonella isolates (S. Typhi from 11 individuals and S. Paratyphi A from 12 individuals) were collected from consecutive patients attending the Institute for Tropical Diseases during the period January 2002 to August 2008. All isolates were derived from positive blood cultures. Isolates were initially identified biochemically, followed by confirmation using specific antisera. Serotype determination was performed at the National Institute for Public Health and the Environment (RIVM) (Bilthoven, The Netherlands).

  A c c e p t e d M a n u s c r i p t 7 by the microbroth dilution technique. All tests were performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines where applicable. For in vitro detection of efflux pump expression, a two-step dilution series of the antibiotics nalidixic acid, ciprofloxacin and chloramphenicol was made to detect the MIC in the absence and presence of the efflux pump inhibitor (EPI) phenylalanine naphthylamide (PAN) (20 g/mL). A reduction of the initial MIC by 3-step difference upon exposure to the EPI is an indication for the presence of an activated efflux pump [7].

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

 8 the manufacturer's instructions. Isolates with 95% identical profiles were considered to be closely related.

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

 10 or reduced susceptibility to fluoroquinolones is due the presence of active efflux pumps, impaired outer membrane permeability or gyrase mutations. As efflux does not explain the current findings, all isolates were subjected to sequencing of the gyrA gene. Mutations in the QRDR region of the gyrA gene resulting in amino acid substitutions are given in Fig.1. All NAL S isolates show no mutations in the QRDR region. The NAL R isolates all had mutations at position 83 resulting in a substitution of serine to phenylalanine or tyrosine.

A

  c c e p t e d M a n u s c r i p t 11 studied, it was not possible to demonstrate the presence of plasmid-encoded qnr genes or aac(6')-Ib-cr, indicating that these qnr-bearing plasmids are 'host' specific.Therefore, these findings confirm other reports that the reduced MICs of ciprofloxacin in S. Typhi and S. Paratyphi A are solely due to the amino acid substitution in the QRDR 'cluster'. Besides reduced susceptibility to quinolones, two S. Typhi isolates demonstrated resistance to ampicillin, chloramphenicol, tetracycline and trimethoprim. One S. Paratyphi A isolate was resistant to ampicillin, chloramphenicol and tetracycline. In the two trimethoprim-resistant S. Typhi isolates an 800-bp integron cassette was demonstrated, indicating that the gene involved is probably the dfrA7 gene encoding for a dihydrofolate reductase[START_REF] Shanahan | Molecular analysis of and identification of antibiotic resistance genes in clinical isolates of Salmonella typhi from India[END_REF]. Integrons have been demonstrated in non-typhoidal Salmonella but only occasionally in S. Typhi[17].

  Tamang MD, Oh JY, Seol SY, Kang HY, Lee JC, Lee YC, et al. Emergence of multidrug-resistant Salmonella enterica serovar Typhi associated with a class 1 integron carrying the dfrA7 gene cassette in Nepal. Int J Antimicrob Agents 2007;30:330-5.

Fig. 1 .

 1 Fig. 1. Genotyping of 23 isolates by repetitive sequence-based polymerase chain reaction (rep-PCR) (DiversiLab ® ). MIC, minimum inhibitory concentration; QRDR,

Funding

None.

Competing interests

None declared.

Ethical approval

Not required.

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


Spyros Pournaras

Evangelia Dimitroulia

Aggeliki Poulou

Georgia Vrioni

Evangelia Neou

John Dendrinos

Athanassios Tsakris

Carbapenemase Kpc

Tigecycline Colistin

Activity of tigecycline alone and in combination with colistin and meropenem against Klebsiella pneumoniae carbapenemase (KPC

Activity of tigecycline alone and in combination with

colistin and meropenem against carbapenemase (KPC)-producing Enterobacteriaceae strains by time-kill assay

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. E-mail address: atsakris@med.uoa.gr (A. Tsakris).

bacteriostatic in five strains. The tigecycline + meropenem combination was not bactericidal against the four K. pneumoniae strains and was non-synergistic against all eight strains. Tigecycline + colistin was bactericidal against all strains at most time intervals and concentrations and was also synergistic at 1 and 2 MIC against most strains up to 4-8 h and at 4 MIC up to 24 h against all strains. These findings suggest that, at most drug concentrations, tigecycline, colistin and meropenem as single agents do not exhibit efficient bactericidal activity against most of the KPC-producing strains. Tigecycline alone might be a therapeutic option for infections caused by KPC-producers when bacteriostatic activity is adequate or combined with colistin when bactericidal activity is necessary.

Additional in vivo tests are warranted to assess better the killing kinetics of tigecycline combinations against KPC-producers.

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

Introduction

Since the beginning of the last decade, Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae have been increasingly detected in the USA and subsequently in several regions worldwide [START_REF] Bratu | Rapid spread of carbapenem-resistant Klebsiella pneumoniae in New York City: a new threat to our antibiotic armamentarium[END_REF][START_REF] Nordmann | The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria[END_REF]. KPC enzymes confer various levels of resistance to all -lactams, including carbapenems [START_REF] Nordmann | The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria[END_REF].

Moreover, bla KPC genes are easily transferable and are often linked with various non -lactam resistance determinants, further compromising the therapeutic alternatives for clinically significant infections [START_REF] Bratu | Rapid spread of carbapenem-resistant Klebsiella pneumoniae in New York City: a new threat to our antibiotic armamentarium[END_REF][START_REF] Nordmann | The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria[END_REF][START_REF] Tsakris | First occurrence of KPC-2-possessing Klebsiella pneumoniae in a Greek hospital and recommendation for detection with boronic acid disc tests[END_REF]. Clinical reports have already documented that hospital infections due to KPC-possessing Enterobacteriaceae are commonly associated with increasing therapeutic failure and mortality [START_REF] Bratu | Rapid spread of carbapenem-resistant Klebsiella pneumoniae in New York City: a new threat to our antibiotic armamentarium[END_REF][START_REF] Nordmann | The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria[END_REF][START_REF] Hirsch | Detection and treatment options for Klebsiella pneumoniae carbapenemases (KPCs): an emerging cause of multidrugresistant infection[END_REF]. Beside these observations, studies dealing with antimicrobial treatment of KPC infections and clinical outcomes are based on a limited number of case patients and therefore the optimum treatment has not been well established [START_REF] Hirsch | Detection and treatment options for Klebsiella pneumoniae carbapenemases (KPCs): an emerging cause of multidrugresistant infection[END_REF]. In this respect, in vitro studies examining antibiotic combinations and well controlled clinical trials are needed to ascertain the optimum treatment of KPC infections.

Susceptibility testing data suggest that treatment of infections caused by KPCproducers commonly requires the use of tigecycline or colistin as last-resort drugs; meropenem in many cases also retains phenotypic activity against KPCproducers and is considered as a possible alternative [START_REF] Nordmann | The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria[END_REF][START_REF] Hirsch | Detection and treatment options for Klebsiella pneumoniae carbapenemases (KPCs): an emerging cause of multidrugresistant infection[END_REF][START_REF] Pournaras | Clonal spread of KPC-2 carbapenemase-producing Klebsiella pneumoniae in Greece[END_REF]. Tigecycline, a glycylcycline antibiotic approved by the US Food and Drug Administration (FDA) for the treatment of complicated intra-abdominal infections and skin and skinstructure infections in adults, has been found to be active against A c c e p t e d M a n u s c r i p t Enterobacteriaceae regardless of the presence of carbapenemases [START_REF] Castanheira | Antimicrobial activities of tigecycline and other broad-spectrum antimicrobials tested against serine carbapenemase-and metallo--lactamase-producing Enterobacteriaceae: report from the SENTRY Antimicrobial Surveillance Program[END_REF]. Its action is mediated by the inhibition of bacterial protein synthesis and is mainly bacteriostatic. The in vitro activity of tigecycline against KPC-producers has been only scarcely tested [START_REF] Nordmann | The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria[END_REF][START_REF] Bratu | Carbapenemase-producing Klebsiella pneumoniae in Brooklyn, NY: molecular A c c e p t e d M a n u s c r i p t epidemiology and in vitro activity of polymyxin B and other agents[END_REF], whilst antibiotic combinations including tigecycline have not been studied previously in these bacteria. To assess further the in vitro efficacy of tigecycline alone or combined with colistin and meropenem, a time-kill study of KPC-producing clinical isolates belonging to several Enterobacteriaceae species was undertaken. The purpose of this study was to provide data regarding effective antibiotics or antibiotic combinations against KPC-producers that could be of value to clinicians treating KPC infections at the bedside.

Materials and methods

Bacterial strains

Susceptibility testing

Tigecycline, colistin and meropenem minimum inhibitory concentrations (MICs) were determined by broth macrodilution [START_REF]Performance standards for antimicrobial susceptibility testing; twentieth informational supplement[END_REF], which is normally applied as a reference method for isolates to be tested by killing curve assays. For tigecycline, the FDA recommendation was used (susceptible, ≤2 g/mL; and resistant, ≥8 g/mL), and for colistin the European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoint for Enterobacteriaceae (susceptible, ≤2 g/mL; and resistant, ≥4 g/mL) were used [START_REF]Breakpoint tables for interpretation of MICs and zone diameters, version 1.0[END_REF]. For meropenem, the Clinical and Laboratory Standards Institute (CLSI) breakpoints modified in June 2010 [START_REF]Performance standards for antimicrobial susceptibility testing; twentieth informational supplement[END_REF] were used. Colistin was not tested against the S. marcescens clinical strain as this species exhibits intrinsic resistance to colistin.

Time-kill curve analyses

Time-kill curves were performed in triplicate by inoculating 5  10 5 colonyforming units (CFU)/mL of the test organisms into 3 mL of fresh cation-adjusted Mueller-Hinton broth. Antibiotics (tigecycline, colistin and meropenem as single agents, tigecycline + colistin and tigecycline + meropenem) were added at concentrations of 1, 2 and 4 MIC for each strain [START_REF] Cobo | Use of tigecycline for the treatment of prolonged bacteremia due to a multiresistant VIM-1 and SHV-12 -lactamase-producing Klebsiella pneumoniae epidemic clone[END_REF]. Aliquots were removed at 0, 2, 4, 6, 8, 16 and 24 h post inoculation, serially diluted and plated on A c c e p t e d M a n u s c r i p t Mueller-Hinton agar plates for enumeration of viable colonies. Antimicrobial carry-over was controlled by widely streaking the transferred aliquot over the agar plate and observing for possible inhibition of growth at the site of the initial streak. Bactericidal activity was defined as a ≥3 log 10 reduction in the total CFU/mL from the original inoculum. Bacteriostatic activity was defined as maintenance of or <3 log 10 reduction in the total CFU/mL from the original inoculum [START_REF]Performance standards for antimicrobial susceptibility testing; twentieth informational supplement[END_REF]. Time-kill curves were constructed by plotting mean colony counts versus time. Synergy was defined as ≥2 log 10 decrease in the number of CFU/mL between the combination and the most active compound. Synergy time-kills are usually read after 24 h of incubation; however, it is believed that assessment of viability at earlier time intervals may also have clinical significance [START_REF]Breakpoint tables for interpretation of MICs and zone diameters, version 1.0[END_REF], as the responsiveness according to the time of exposure may represent the dosage intervals used in patients. Thus, all time periods were evaluated. Indifference was defined at 24 h as a ±1 log 10 kill to <2 log 10 compared with the most efficient agent alone [START_REF] Vidaillac | In vitro activity of ceftaroline alone and in combination against clinical isolates of resistant Gram-negative pathogens, including -lactamase-producing Enterobacteriaceae and Pseudomonas aeruginosa[END_REF].

Results

Macrodilution MICs were 0.25-4 g/mL for tigecycline, 0.5-1 g/mL for colistin and 2-16 g/mL for meropenem (Table 1). Using previous CLSI criteria, four strains were considered as susceptible to meropenem, having MICs of 2-4 g/mL, but by applying the updated CLSI criteria [START_REF]Performance standards for antimicrobial susceptibility testing; twentieth informational supplement[END_REF] all strains were classified as non-susceptible. The mean log 10 CFU/mL changes from the initial bacterial A c c e p t e d M a n u s c r i p t concentrations with the antibiotics tested as single agents and in combinations during the time-kill assays are presented in Table 1.

Tigecycline alone displayed a concentration-independent bacteriostatic effect in seven strains, with moderate re-growth after 8-16 h of incubation. In the remaining one K. pneumoniae strain, tigecycline was bactericidal at 16 h, with subsequent re-growth. Meropenem alone had a concentration-independent bactericidal activity at 24 h in three strains (one each of E. coli, E. cloacae and S. marcescens). It is of note that in both E. coli strains with low meropenem MICs the responsiveness to meropenem varied considerably at 1 and 2 MIC, with the one strain being effectively killed and the second strain remaining unaffected at 24 h, implying a potential for heteroresistance. In the four K. pneumoniae strains, bacteriostatic activity was maintained for 8 h, with significant re-growth observed. Colistin as a single agent showed bactericidal activity at 8-16 h in only three of the seven strains tested (one each of E. coli, K. pneumoniae and E. cloacae) only at 4 MIC. Furthermore, consistent re-growth was observed in these strains beyond 8-16 h, causing a loss of bactericidal activity. For the remaining clinical strains and concentrations used, colistin caused a bacteriostatic effect, with re-growth after 6-8 h. Tigecycline + colistin exerted bactericidal activity against all strains at most time intervals and concentrations tested, although a degree of re-growth was observed in some strains at 16-24 h. In addition, this combination was A c c e p t e d M a n u s c r i p t synergistic at 1 and 2 MIC against most organisms at 4 h and 8 h, and at 4 MIC the synergism was sustained at 24 h against all strains compared with either tigecycline or colistin alone. In contrast, tigecycline + meropenem was nonsynergistic against all strains and was also not bactericidal against the four K. pneumoniae strains. The non-KPC-producing control strain did not exhibit significant differences in its responsiveness to the antibiotics tested compared with the study strains. The time-kill profiles obtained with 4 MIC of tigecycline, colistin and tigecycline + colistin are presented in Fig. 1.

Discussion

KPC-producers cause severe hospital infections, commonly requiring combination therapies [START_REF] Nordmann | The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria[END_REF][START_REF] Hirsch | Detection and treatment options for Klebsiella pneumoniae carbapenemases (KPCs): an emerging cause of multidrugresistant infection[END_REF][START_REF] Pournaras | Clonal spread of KPC-2 carbapenemase-producing Klebsiella pneumoniae in Greece[END_REF][START_REF] Bratu | Carbapenemase-producing Klebsiella pneumoniae in Brooklyn, NY: molecular A c c e p t e d M a n u s c r i p t epidemiology and in vitro activity of polymyxin B and other agents[END_REF]. However, there is a lack of information regarding the possible bactericidal activity of existing antimicrobials, alone or in combination, against KPC-producing bacteria.

The results of the present study have shown that, at most drug concentrations tested, tigecycline, colistin and meropenem as single agents do not exhibit efficient bactericidal activity against KPC-producing Enterobacteriaceae. Similar to these findings, previous reports have shown that tigecycline has bacteriostatic activity against Enterobacteriaceae exhibiting resistance to several unrelated antimicrobial classes [START_REF] Pankey | In vitro antibacterial activity of tigecycline against resistant Gram-negative bacilli and enterococci by time-kill assay[END_REF][START_REF] Zhanel | Determination of the pharmacodynamic activity of clinically achievable tigecycline serum concentrations against clinical isolates of Escherichia coli M a n u s c r i p t with extended-spectrum -lactamases, AmpC -lactamases and reduced susceptibility to carbapenems using an in vitro model[END_REF]. A single study testing tigecycline alone against KPC-producing K. pneumoniae isolates also showed bacteriostatic activity [START_REF] Bratu | Carbapenemase-producing Klebsiella pneumoniae in Brooklyn, NY: molecular A c c e p t e d M a n u s c r i p t epidemiology and in vitro activity of polymyxin B and other agents[END_REF], whilst similar results have been exerted by tigecycline against one metallo--A c c e p t e d M a n u s c r i p t lactamase-producing K. pneumoniae isolate [START_REF] Cobo | Use of tigecycline for the treatment of prolonged bacteremia due to a multiresistant VIM-1 and SHV-12 -lactamase-producing Klebsiella pneumoniae epidemic clone[END_REF]. Colistin has also been found to exert concentration-dependent bactericidal activity against KPC-producing K.

pneumoniae isolates [START_REF] Bratu | Carbapenemase-producing Klebsiella pneumoniae in Brooklyn, NY: molecular A c c e p t e d M a n u s c r i p t epidemiology and in vitro activity of polymyxin B and other agents[END_REF]. However, in the current study colistin exposure was followed by re-growth in all strains, including those exhibiting an initial bactericidal activity, possibly explaining why colistin when used alone commonly exhibits low clinical success rates [START_REF] Hirsch | Detection and treatment options for Klebsiella pneumoniae carbapenemases (KPCs): an emerging cause of multidrugresistant infection[END_REF]. However, the latter literature review [START_REF] Hirsch | Detection and treatment options for Klebsiella pneumoniae carbapenemases (KPCs): an emerging cause of multidrugresistant infection[END_REF] revealed that the outcomes were improved when colistin was combined with other antibiotics and this is also suggested by the time-kill experiments in this study. With regard to meropenem, its bactericidal activity against only three of the eight strains tested supports the clinical data of carbapenem-susceptible KPC infections treated by carbapenem monotherapy, which frequently leads to clinical and microbiological failures, being successful in only 40% of cases [START_REF] Hirsch | Detection and treatment options for Klebsiella pneumoniae carbapenemases (KPCs): an emerging cause of multidrugresistant infection[END_REF][START_REF] Weisenberg | Clinical outcomes of patients with Klebsiella pneumoniae carbapenemase-producing K. pneumoniae after treatment with imipenem or meropenem[END_REF].

The in vitro effect of tigecycline combined with other agents against KPCproducers has not been tested previously. The present study has shown a lack of synergism using the combination of tigecycline + meropenem. It should be noted that this combination is usually considered as a therapeutic alternative against multidrug-resistant Gram-negative bacilli. In contrast, the combination of tigecycline + colistin was found here to be bactericidal against all strains. This result supports previous findings that this combination was bactericidal against K.

pneumoniae isolates [START_REF] Entenza | Tigecycline in combination with other antimicrobials: a review of in vitro, animal and case report studies[END_REF] as well as initial clinical observations that tigecycline + colistin succeeded against most KPC infections [START_REF] Hirsch | Detection and treatment options for Klebsiella pneumoniae carbapenemases (KPCs): an emerging cause of multidrugresistant infection[END_REF]. It was also synergistic at 1 and 2 MIC at 4 h and 8 h for most organisms tested, whilst synergy was CFU, colony-forming units; N/A, not applicable (as S. marcescens is intrinsically resistant to colistin).

a <3 log 10 reduction in CFU, implies bacteriostatic effect.

  author. Tel.: +30 210 746 2011; fax: +30 210 746 2210.

  The strains tested comprised eight genetically confirmed KPC-producing enterobacterial clinical strains (four K. pneumoniae, two Escherichia coli, one Enterobacter cloacae and one Serratia marcescens) recovered from patients hospitalised in five tertiary care hospitals located in four Greek regions (two hospitals in the broad region of Athens and one hospital each in Thessaloniki, Larissa and Serres). Polymerase chain reaction (PCR) and sequencing assays showed that all microorganisms produced KPC-2 carbapenemase. The K. pneumoniae and E. coli strains were randomly selected among those representing different clonal types of KPC-possessing pathogens. The non-A c c e p t e d M a n u s c r i p t carbapenemase-producing E. coli strain ATCC 25922 was used as a control in all assays.

A

  c c e p t e d M a n u s c r i p t achieved at multiple time points and was sustained after 24 h at 4 MIC, although such a concentration is hardly achievable in serum. The results of this study indicate that tigecycline combined with colistin at appropriate dosage intervals might be a therapeutic option for infections due to multidrug-resistant KPCproducers when bactericidal activity is necessary, such as in bacteraemia, endocarditis or other severe infections. However, additional in vivo tests are warranted to assess better the performance of tigecycline combinations with antibiotics that may have different pharmacokinetic and pharmacodynamic parameters against KPC infections.

Fig. 1 .

 1 Fig. 1. Time-kill kinetics performed in fresh cation-adjusted Mueller-Hinton broth

Table 1

 1 Tigecycline, meropenem and colistin minimum inhibitory concentrations (MICs) of the tested strains and mean changes from initial bacterial concentrations following incubation for 24 h with tigecycline, colistin, meropenem, tigecycline + colistin and tigecycline + meropenem during time-kill assays at 1, 2 and 4 MIC

	Antimicrobial	Escherichia	E.	Enterobacter	Serratia	Klebsiella	K.	K.	K.	E. coli
	agent/combination	coli 1	coli	cloacae	marcescens	pneumoniae	pneumoniae	pneumoniae	pneumoniae	ATCC
			2			1	2	3	4	25922
	Tigecycline MIC	0.5	0.25 1	4	0.5	1	1	2	0.25
	(g/mL)
	Mean change (log 10 CFU/mL)
	1 MIC	-1	-2.2	-0.9	-1.3	-0.6	-1.6	-1.7	-2	-0.4
			a
	2 MIC	0	0	0	-0.2	0	0	-2.9	-1.6	-1.6
	4 MIC	-1.8	-0.7 -1.3	-0.5	-0.6	-0.7	-1.8	-0.7	-2.1
	Meropenem MIC	2	2	16	4	4	16	16	8	0.06
	(g/mL)
	Mean change (log 10 CFU/mL)
	1 MIC	-3.5 b	0	-3 b	-2.8	0	0	0	-0.3	-5.6 b
	2 MIC	-5.6 b	0	-5.6 b	-3.3 b	0	0	0	0.6	-5.6 b
	4 MIC	-4.3 b	-2.9 -2.9	-3.6 b	-0.6	-0.2	0	0	-5.6 b

Funding

None.

Competing interests

None declared.

Ethical approval

Not required. Edited Figure 1


Katrin Reder-Christ

Hildegard Falkenstein-Paul

Gabriela Klocek

Saad Al- Kaddah

Udo Bakowsky

Gerd Bendas

Model membrane approaches to determine the role of calcium for the antimicrobial activity of friulimicin

Keywords:

Friulimicin is a cyclic lipopeptide antibiotic, currently in clinical development, that possesses excellent activity against Gram-positive bacteria, including multiresistant strains. A recent study on the mode of action of friulimicin reported on the interference with bacterial cell wall biosynthesis via a calcium-dependent complexing of the bactoprenol phosphate carrier C 55 -P. The calcium dependency of this noncommon targeted activity remains to be elucidated. In the present model membrane approach, the role of calcium for friulimicin targeting to C 55 -P was investigated by biosensor-based detection of binding affinities. The findings were supplemented by atomic force microscopy (AFM) and circular dichroism (CD) spectroscopy.

Comparing the calcium salt of friulimicin with the calcium-free peptide, calcium appeared to be essential for friulimicin interaction with 1,2-dioleoyl-sn-glycero-3phosphocholine (DOPC) model membranes. The binding affinity was even higher in the presence of 0.1 mol% C 55 -P (0.21 M vs. 1.22 M), confirming the targeted mode of action. Binding experiments with supplemented calcium salts suggest (i) the phosphate group as the essential moiety of C 55 -P, referring to a bridging function of calcium between the negatively charged friulimicin and C 55 -P, and (ii) a structural effect of calcium shifting the peptide into a suitable binding conformation (CD spectra). AFM images confirmed that calcium has no, or only a minor, effect on the aggregate formation of friulimicin. These data shed new light on the mechanisms of antibacterial activity of friulimicin.

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

Introduction

Friulimicin is a cyclic lipopeptide antibiotic isolated from Actinoplanes friuliensis [START_REF] Vértesy | Friulimicins: novel lipopeptide antibiotics with peptidoglycan synthesis inhibiting activity from Actinoplanes friuliensis sp. nov. II. Isolation and structural characterization[END_REF] and is active against many Gram-positive bacteria, including antibiotic-resistant strains, e.g. meticillin-resistant Staphylococcus aureus (MRSA) [START_REF] Bemer | In vitro activities of a new lipopeptide, HMR 1043, against susceptible and resistant Gram-positive isolates[END_REF]. It is currently in clinical development and is a promising drug candidate. Friulimicin consists of a cyclic decapeptide that is N-terminally linked to an unsaturated branched fatty acid (Fig. 1A) [START_REF] Vértesy | Friulimicins: novel lipopeptide antibiotics with peptidoglycan synthesis inhibiting activity from Actinoplanes friuliensis sp. nov. II. Isolation and structural characterization[END_REF].

Despite variations in the length and branch of the fatty acids as well as in the number of amino acid residues, all cyclic lipopeptides share amphipathic characteristics. The lipid side chain and hydrophobic amino acid residues are balanced by the overall net negative charge of the lipopeptides, making them highly soluble in aqueous solution [START_REF] Baltz | Natural products to drugs: daptomycin and related lipopeptide antibiotics[END_REF]. The macrocycle possesses three amino acid residues that are conserved in all cyclic lipopeptides and probably show a similar conformation that might be important for their activity.

However, the mode of action of lipopeptides has not been fully elucidated so far.

Most studies have been done with daptomycin, which received US Food and Drug Administration (FDA) approval in 2003. Its proposed mode of action derives from the interaction with bacterial membranes. Daptomycin aggregates in the presence of calcium. The resulting conformational changes induce an interaction with the bacterial membrane, a reduction of the membrane potential, leakage and cell death [START_REF] Silverman | Correlation of daptomycin bactericidal activity and membrane depolarization in Staphylococcus aureus[END_REF][START_REF] Straus | Mode of action of the new antibiotic for Gram-positive pathogens daptomycin: comparison with cationic antimicrobial peptides and lipopeptides[END_REF]. In recent studies, daptomycin was assumed to have an impact on the cell envelope integrity. It even might affect the lipid II cycle [START_REF] Wecke | Daptomycin versus friulimicin B: in-depth profiling of Bacillus subtilis cell envelope stress responses[END_REF].

A c c e p t e d M a n u s c r i p t 5 Despite its structural similarity to daptomycin, friulimicin has a different mode of action. Schneider et al. [START_REF] Schneider | The lipopeptide antibiotic friulimicin B inhibits cell wall biosynthesis through complex formation with bactoprenol phosphate[END_REF] recently showed that friulimicin complexes the bactoprenol phosphate carrier C 55 -P in a calcium-dependent manner and thus inhibits cell wall biosynthesis. Membrane integrity was not influenced [START_REF] Schneider | The lipopeptide antibiotic friulimicin B inhibits cell wall biosynthesis through complex formation with bactoprenol phosphate[END_REF]. Although the calciumdependency of friulimicin activity was shown, the role of calcium ions has yet to be clarified.

The present model membrane approach affords insights into the role of calcium in the mode of action of friulimicin and presents more details on targeting C 55 -P.

Detection of kinetic constants of friulimicin binding to C 55 -P in model membranes by quartz crystal microbalance (QCM) confirms that (i) the phosphate is the crucial moiety of the target structure C 55 -P and (ii) calcium is essential for friulimicin binding to C 55 -P. Atomic force microscopy (AFM) and circular dichroism (CD) spectroscopy show that calcium has no, or only a minor, effect on peptide aggregate formation, but mediates a favourable conformation of the peptide molecules for binding C 55 -P. These data reflect the results of Schneider et al. [START_REF] Schneider | The lipopeptide antibiotic friulimicin B inhibits cell wall biosynthesis through complex formation with bactoprenol phosphate[END_REF] referring to C 55 -P as the friulimicin target and, furthermore, shed new light on the processes at the bacterial membrane surface.

Materials and methods

Chemicals

All commercial reagents were of highest quality and were used without further purification. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) was purchased from Avanti Polar Lipids Inc. (Alabaster, AL). C 55 -P (undecaprenyl phosphate) was A c c e p t e d M a n u s c r i p t 6 purchased from Larodan Fine Chemicals AB (Malmö, Sweden). Dicetylphosphate (DCP) was from Sigma (Deisenhofen, Germany). Friulimicin B (FRI) and calcium chloride-associated friulimicin B (FRI-CA) were kindly provided by Prof. Hans-Georg Sahl (Universität-Bonn, Bonn, Germany).

Model membrane preparation

Cleaning and preparation procedures of the quartz sensors for QCM have been described previously [START_REF] Christ | The detection of UVinduced membrane damages by a combination of two biosensor techniques[END_REF]. Preparation of microscope glass slides for AFM were described [START_REF] Christ | The role of lipid II in membrane binding of and pore formation by nisin analyzed by two combined biosensor techniques[END_REF]. For both QCM and AFM, the supported bilayers were completed by transferring a monolayer consisting of either pure DOPC, DOPC/0.1 mol% C 55 -P or DOPC/0.1 mol% DCP by Langmuir-Blodgett technique onto the covalently fixed first monolayer.

Quartz crystal microbalance experiments

QCM experiments were performed at 25 C using a LiquiLab21 system (ifak e.V., Barleben, Germany). After reaching a constant frequency, 3 mL of a 1 M peptide solution was injected into the flow system. The run was stopped after reaching a constant frequency. Binding constants were calculated from the frequency traces as described previously [START_REF] Christ | The role of lipid II in membrane binding of and pore formation by nisin analyzed by two combined biosensor techniques[END_REF].

Atomic force microscopy experiments

AFM images were obtained on a NanoWizard ® (JPK Instruments, Berlin, Germany) and on a Digital NanoScope IV BioScope (Veeco Instruments, Santa Barbara, CA).

Details have been described previously [START_REF] Christ | The role of lipid II in membrane binding of and pore formation by nisin analyzed by two combined biosensor techniques[END_REF].

A c c e p t e d

M a n u s c r i p t 7

Circular dichroism spectroscopy

CD measurements of FRI, FRI-CA and pre-incubated friulimicin (FRI-PI) were taken at 25 C using a Chirascan TM CD spectrometer (Applied Photophysics Ltd., Leatherhead, UK). A quartz cuvette with a path length of 0.1 cm was used. Scans were obtained at 0.5 nm resolution, 1 nm bandwidth and at a scan speed of 7 s per point. All spectra were measured in water and were corrected by subtracting the baseline. Results are reported as mean residue ellipticity in units of deg cm 2 dmol -1 .

Results and discussion

Friulimicin binding depends on C55-P and peptide-associated calcium

Binding to C 55 -P is not a common mode of action of antibiotics and has only been described so far for friulimicin. To confirm further this target process, the binding kinetics of friulimicin to DOPC ± 0.1 mol% C 55 -P model membranes using a QCM biosensor was analysed. Although bacterial membranes contain a certain fraction of negatively charged lipids, the non-charged DOPC was consciously selected as the matrix lipid to focus on the role of calcium in the interaction of the negatively charged friulimicin with C 55 -P and to avoid a charge-induced calcium accumulation at the membrane surface. Friulimicin was used either as calcium-free peptide (FRI) or associated with calcium chloride (FRI-CA) with two calcium ions per peptide molecule. The role of C 55 -P as the FRI-CA target could be confirmed. The binding affinity of FRI-CA to DOPC/C 55 -P was nearly six-fold higher than to pure DOPC membranes (k D = 0.21 M vs. 1.22 M), reflecting the recent findings on the role of C 55 -P as the target for interference with cell wall biosynthesis. The FRI-CA association remains unaffected by the presence of C 55 -P. Thus, the overall better binding to DOPC/C 55 -P is mainly due to a nearly five-fold decelerated dissociation rate (k diss 0.75E -3 s -1 vs.

3.57E -3 s -1 ). Interestingly, this excess of calcium did not improve FRI-CA binding; it even slightly reduced the membrane affinity (not shown). Thus, a peptide-calcium ratio of 1:2, as in the case of FRI-CA, appears sufficient to mediate peptide binding. Further calcium ions appear to affect the overall peptide aggregation or directly influence the membrane.

The phosphate moiety of C55-P dominates friulimicin binding

However, the inability of FRI to bind to DOPC/C 55 P turned to a moderate binding affinity in presence of 1.25 mM CaCl 2 solution [k D = 1.9 ± 1.25 M; association rate (k ass ) = 1138 ± 635 M -1 s -1 ; k diss = 1.18E -3 ± 0.90E -3 s -1 ]. Although this affinity is lower than that of FRI-CA, it indicates that the contact of FRI with Ca 2+ has an evident effect on peptide binding. Different functions of calcium in this process were conceivable. Calcium could mediate the binding of the negatively charged binding partners or could directly affect the peptide and thus enable binding. attain the levels of FRI-CA, which was mainly caused by a diminished dissociation rate k diss for FRI-CA. However, it became evident that calcium affects the peptide before binding to C 55 -P, e.g. by mediating the peptide-peptide interaction or by influencing the conformation of the peptide molecules, thus making them accessible for C 55 -P binding. Both possibilities are discussed below.

Role of calcium for peptide-peptide interaction

AFM was used to focus on the role of calcium for peptide aggregation during membrane contact. DOPC forms even and homogeneous membranes (Fig. 2A, left).

Addition of FRI or FRI-CA provided identical results. As shown for FRI-CA, both peptides did not influence the DOPC layer but formed giant aggregates of dimension ca. 50 nm (Fig. 2A, right) that appear to be loosely attached at the membrane and do not reflect the different binding characteristics of FRI and FRI-CA. High-performance liquid chromatography (HPLC) data further confirmed the similarity of the peptide aggregates of FRI and FRI-CA according to their nearly identical elution profiles (30 g peptide/50 g buffer) (not shown).

Calcium influences the secondary structure of friulimicin

Since FRI and FRI-CA formed giant aggregates of comparable dimensions, calcium obviously has only a minor influence on peptide aggregation. It might rather influence the conformation of the peptide molecules thus making them accessible for C 55 -P binding. CD measurements were performed using peptide concentrations of 75 M interaction. It became evident that the spectra of FRI, FRI-CA and FRI-PI do not resemble the spectra of conventional secondary structures such as the -helix, sheet, -turn or random coil (Fig. 2B). It seems that the friulimicin spectrum is a superposition of two types of secondary structures, but at this stage of the experiments we can neither confirm nor exclude it comprehensively.

The interaction with calcium caused a slight shift (2-3 nm) of the two maxima (194 nm and 213 nm) towards lower wavelengths. Furthermore, an increase in the amplitude of the signal is observed for the 75 M preparation. Both effects suggest a stabilisation of the peptide secondary structure. Such an increase in the amplitude of the negative CD signal upon addition of Ca 2+ could also be observed for troponin C, reflecting increased stability of the helical regions [START_REF] Smith | The effects of deletion of the amino-terminal helix on troponin C function and stability[END_REF]. Similar findings were reported for daptomycin, where addition of CaCl 2 induced an increase in the positive ellipticity maximum and a slight shift from 233 nm to 231 nm [START_REF] Jung | Structural transitions as determinants of the action of the calcium-dependent antibiotic daptomycin[END_REF]. However, calcium was also shown to affect daptomycin aggregation [START_REF] Ho | Effect of divalent cations on the structure of the antibiotic daptomycin[END_REF]. Although the spectra suggest a friulimicin aggregation with increasing peptide concentrations (decreased amplitude for 150 M), the impact of calcium on this process appears to be negligible. Further CD measurements using a series of friulimicin concentrations confirm that aggregation is mainly a concentration-triggered process (not shown). Nevertheless, the indicated shift in the maxima in the presence of calcium was evident at all concentrations used, especially that at 213 nm. An impact of calcium in mediating a stabilised conformation of friulimicin can thus be postulated and reflects the QCM binding studies.

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

This was also confirmed by AFM images. Compared with pure DOPC membranes (Fig. 2A, left), DOPC/C 55 -P membranes show protruding structures (Fig. 3A), suggesting a clustered arrangement of C 55 -P within the membrane. As observed for DOPC membranes, FRI attached in an aggregated form to the DOPC/C 55 -P membranes (Fig. 3B). However, these aggregates appear to be more evenly distributed as a 4-5 nm layer. It might be assumed that the FRI aggregates accumulate in the areas of the C 55 -P clusters, leading to an even peptide distribution.

Nevertheless, this should not be based on a specific recognition of FRI. These aggregates are not the sensitive structures for binding and the density of the layer contradicts a monomolecular association of the peptides to the membrane. FRI-CA binds at the C 55 -P containing membrane as a thin and dense layer with a detectable surface roughness below 1 nm (Fig. 3C). No aggregates were detectable.

In some areas, height differences of ca. 1 nm appeared, which should correspond to the surface structures of the DOPC/C 55 -P membranes. The image refers to a tight peptide binding at the membrane and thus emphasises the present findings on the necessity of calcium and C 55 -P as the target structure for friulimicin membrane interaction.

Conclusion

The present model membrane approach gives new insights into the antibiotic mode of action of the lipopeptide friulimicin. C 55 -P was clearly confirmed as the target structure by kinetic binding data using QCM. The phosphate moiety of C 55 -P appeared as the essential structure for friulimicin binding. Divalent calcium is essential for this binding process and acts as a bridge between C 55 -P and friulimicin.

Fig

  Fig.1Billustrates the binding kinetics of both peptides in aqueous solution. In

  QCM data confirmed the target function of C 55 -P and the general need of calcium for friulimicin binding. The role of calcium might be explained by a bridging function between the negatively charged peptide and the phosphate moiety of C 55 -P. To focus further on the role of phosphate, C 55 -P was replaced by dicetylphosphate (DCP), which also possesses a phosphate moiety at the membrane surface but is membrane-anchored by two hexadecylalkane chains. Nearly identical binding affinities of FRI-CA to DOPC/C 55 -P and DOPC/DCP could be obtained (k D = 0.21 ± 0.08 M and 0.28 ± 0.07 M, respectively), suggesting the dominant role of the phosphate moiety. The association and dissociation rates of both were also in good agreement (not shown). An active role of the bactoprenol moiety for friulimicin binding can be excluded, although the relative high binding affinity of FRI-CA to pure DOPC membranes might indicate unspecific interactions with the membrane, most likely via the lipid chain of the peptide.

A c c e p t e d M a n u s c r i p t 9 3. 3 .

 93 Role of calcium in the friulimicin binding processSince calcium was shown to be essential for friulimicin binding, one might assume that additional calcium ions would further increase binding. Therefore, FRI and FRI-CA binding to DOPC ± 0.1 mol% C 55 P was analysed in the presence of physiological calcium concentrations (1.25 mM CaCl 2 solution).

10 (

 10 The latter became evident by pre-incubation experiments. FRI was pre-incubated in 1.25 mM calcium solution overnight. The binding experiments were subsequently performed in pure aqueous solution. Consequently, remaining calcium had to be in a peptide-associated form. Fig.1Cdisplays the binding data of pre-incubated friulimicin A c c e p t e d M a n u s c r i p t FRI-PI) to DOPC/C 55 -P compared with FRI-CA. The association rate k ass of FRI-PI reached nearly the level of FRI-CA. Overall binding of FRI-PI did not completely

A

  c c e p t e d M a n u s c r i p t 11 and 150 M to determine changes in the secondary structure of friulimicin upon Ca 2+

A

  c c e p t e d M a n u s c r i p t 13 Further focus on the role of calcium in the binding process suggests that calcium has no, or only a minor, effect on the peptide aggregation (AFM images). Nevertheless, CD spectra indicate that calcium indeed influences the conformation of the peptide molecules and obviously shifts the peptide into a suitable form for target binding.Altogether, the model membrane approach in combination with different analytical tools allow for the first time an insight into the distinct functions of calcium in the antimicrobial activity of friulimicin.

Fig. 1 .

 1 Fig. 1. (A) Structure of friulimicin. A cyclic decapeptide is N-terminally linked to an

Fig. 2 .

 2 Fig. 2. (A) Atomic force microscopy images to illustrate the interaction of friulimicin

Fig. 3 .

 3 Fig. 3. Atomic force microscopy images to illustrate the binding of friulimicin to 1,2-

Acknowledgments

The authors would like to thank Prof. Hans-Georg Sahl for providing the friulimicin peptides. Michaele Joosten is acknowledged for performing the HPLC studies.

Funding

This work was supported by the German Research Foundation (FOR 854, Be 2242/3-1).

Competing interests

None declared.

Ethical approval

Not required.