Leonard Amaral
email: lamaral@ihmt.unl.pt

Martin Boeree

Stephen H Gillespie

Zarir F Udwadia

Dick Van Soolingen

Thioridazine cures extensively drug-resistant (XDR-TB) and the need for global trials is now!

Keywords:

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

Introduction

Pulmonary TB is an infectious disease caused by Mycobacterium tuberculosis, which by its co-evolution with man became a steadfast human pathogen when man converted from a hunting-gathering animal to a land-dependent primate.

The prevalence and impact of TB is exacerbated by poverty, overcrowding, famine, war and disease. More recently, diseases that reduce the effectiveness of the immune system, such as human immunodeficiency virus (HIV) infection, predispose the patient to progress from infection to active disease status. In the post-World War II years, especially in the late 1950s, progress in living conditions in the Western world and the introduction of antituberculous drugs fed the dream that TB could finally be eliminated. Progress was made and the number of new TB cases dwindled to a few per hundred thousand persons in most high income nations. In most low income settings, especially those blighted by war or famine, TB remained unchecked. Although the downward trend among the European-or US-born continued, TB among foreign-born individuals increased significantly during the 1970s, reaching still higher levels during the 1980s, 1990s and 2000s [START_REF] Faustini | Risk factors for multidrug resistant tuberculosis in Europe: a systematic review[END_REF]. At first, although TB was found almost exclusively in the migrant population, it soon also increased among nationals, especially those who were co-infected with HIV and presented symptoms of acquired immune deficiency syndrome (AIDS).

By 1992, the scale of new cases of TB infection noted for the city of New York had quadrupled, and of greater alarm was that more than one-half of these new cases of active TB were resistant to the two most effective drugs, isoniazid (INH) and rifampicin (RIF), defined as multidrug-resistant TB (MDR-TB) [START_REF] Frieden | The emergence of drug-resistant tuberculosis in New York City[END_REF]. By the A c c e p t e d M a n u s c r i p t middle of the 1990s, similar observations were made for each of the main urban centres of Europe [START_REF] Faustini | Risk factors for multidrug resistant tuberculosis in Europe: a systematic review[END_REF], although the magnitude of the problem was far less than that experienced in New York City. With huge financial support, the TB control programmes developed in New York took care of the problem within 3 years and the incidence of new cases of active TB was reduced to a lower level than recorded in the 1950s [START_REF] Munsiff | Molecular epidemiology of multidrug-resistant tuberculosis, New York City, 1995-1997[END_REF]. In contrast, in other countries the rates of MDR-TB among new cases continued to increase and the spectrum of drugs to which the strains were resistant also increased [START_REF]Anti-tuberculosis drug resistance in the world[END_REF]. In 2006, the term 'extensively drugresistant TB' (XDR-TB) was introduced to define resistance to INH and RIF (i.e. MDR) and additionally to any fluoroquinolone and at least one of the three injectable antituberculous drugs amikacin, kanamycin and capreomycin [START_REF]Anti-tuberculosis drug resistance in the world[END_REF].

Retrospective analysis of MDR strains from Portugal showed that >50% of all MDR strains are in fact XDR [START_REF] Perdigão | Multidrugresistant tuberculosis in Lisbon, Portugal: a molecular epidemiological perspective[END_REF]. Data from India reveals that ca. 10% of MDR-TB strains are XDR. In Europe, ca. 10% of the 2500 MDR-TB cases occurring in the period 2003-2006 in fact represented XDR-TB [START_REF]Anti-tuberculosis drug resistance in the world[END_REF], but this was strongly associated with former Soviet Union States. These data suggest that the problem of XDR-TB is long-standing but has been unrecognised until the publicity associated with an outbreak of XDR-TB in South Africa, mainly among HIVpositive individuals. Although the extent of the XDR-TB problem is unknown, anecdotal evidence suggests that significant problems exist in all urban centres of Europe, most acutely in some Eastern European countries and in India [START_REF]Anti-tuberculosis drug resistance in the world[END_REF].

Recently, Velayati et al. [START_REF] Velayati | Emergence of new forms of totally drug-resistant tuberculosis bacilli: super extensively drug-resistant tuberculosis or totally drug-resistant strains in Iran[END_REF] reported from Iran new forms of totally drug-resistant TB and coined the phrase 'super extensively drug-resistant tuberculosis'.

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

If the healthcare community is to be successful in controlling this threat, we urgently need better tools for diagnosis and treatment as an effective vaccine is still far in the future. To support the efforts of TB control programmes we must shorten the duration of treatment. Shorter regimens may improve adherence and completion rates, reducing the opportunity for acquisition of new resistance.

However, therapy of the MDR-TB patient is problematic and even with the best possible support [laboratory, directly observed treatment, short-course (DOTS), etc.] and regardless of aggressive therapy involving at least seven or eight drugs, mortality is significant (ca. 15-20%). This is considerably higher if the MDR-TB patient is co-infected with HIV and presents with AIDS. Therapy of the XDR-TB patient is even more difficult. There remains an urgent unmet need for new drugs to manage existing patients with MDR-and XDR-TB. The progress that has been made since the Cape Town Declaration is promising and a TB drug pipeline exists, but the rate at which new molecules will trickle down means that progress will come too late for the hundreds of thousands of current patients struggling to survive their XDR-TB. Moreover, at the time of writing, no clinical trials for therapy of XDR-TB with new agents are taking place (http://clinicaltrials.gov/ct2/results?term=XDR+Tuberculosis). Consequently, there is no completely effective therapy available, apart from the one that we will describe in this article.

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

Thioridazine (TDZ): a promising development

TDZ, a neuroleptic used for the therapy of psychosis, has been safely in use for over four decades. TDZ is an example of a drug that has passed beyond patent protection and is of little interest to the pharmaceutical industry for future development since it offers no promise of financial reward. TDZ has been shown to inhibit in vitro growth of all M. tuberculosis strains studied to date regardless of their antibiotic susceptibility profile [START_REF] Amaral | Inhibition of the respiration of multi-drug resistant clinical isolates of Mycobacterium tuberculosis by thioridazine: potential use for the initial therapy of freshly diagnosed tuberculosis[END_REF]. TDZ enhances the killing of intracellular M.

tuberculosis by non-killing human macrophages including susceptible, MDR and XDR strains [START_REF] Ordway | Clinical concentrations of thioridazine kill intracellular multidrug-resistant Mycobacterium tuberculosis[END_REF][START_REF] Martins | SILA 421, an inhibitor of efflux pumps of cancer cells, enhances the killing of intracellular extensively drug-resistant tuberculosis (XDR-TB)[END_REF]. TDZ cures the mouse of infections with antibiotic-susceptible [START_REF] Martins | The curative activity of thioridazine on mice infected with Mycobacterium tuberculosis[END_REF] and MDR (unpublished data) strains.

TDZ was used for the therapy of XDR-TB patients who did not respond to any antibiotic therapy and whose prognosis was poor and hence were selected for TDZ therapy on the basis of compassionate reasons [START_REF] Abbate | Tuberculosis extensamente resistente (XDR-TB) en Argentina: aspectos destacbles epidemiologicos, bacteriologicos, terapeuticos y evolutivos[END_REF]. As predicted, TDZ cured 10 of 12 XDR-TB patients and the remaining 2 patients responded favourably but dropped out of the programme [START_REF] Abbate | Tuberculosis extensamente resistente (XDR-TB) en Argentina: aspectos destacbles epidemiologicos, bacteriologicos, terapeuticos y evolutivos[END_REF]. However, as is the case for any neuroleptic [START_REF] Thanacoody | Thioridazine: resurrection as an antimicrobial agent?[END_REF], there are patients whose QTc interval is significantly increased thereby placing the patient at risk for sudden death [START_REF] Thanacoody | Thioridazine: resurrection as an antimicrobial agent?[END_REF]. Because patients can be screened with cardiac monitoring prior to and during initial therapy with TDZ, the problem can be minimised and even avoided.

TDZ is ready for global clinical evaluation. It is the only effective drug that has been studied for its antituberculous properties in a systematic fashion-from its A c c e p t e d M a n u s c r i p t activity within a test tube culture to its activity at the site where M. tuberculosis resides, namely the macrophage and its curative activity against antibioticresistant pulmonary TB infections of the mouse and human. TDZ is ready for global clinical trials if only because it is ready for deployment now and may prolong the life of or cure tens of thousands who are infected with a terminal XDR-TB infection.

Compassionate therapy of extensively drug-resistant tuberculosis in India with thioridazine

India bears the burden of most of the worlds MDR-TB. In the latest global drug resistance report [START_REF] Munsiff | Molecular epidemiology of multidrug-resistant tuberculosis, New York City, 1995-1997[END_REF], India accounted for one-third of the world's MDR-TB cases.

There is no public provision for treating these patients who visit multiple public and private medical practitioners in a desperate attempt to treat their drugresistant TB, most serving merely to amplify their resistance further. A huge pool of chronic drug-resistant patients is created and it is not uncommon to see patients who are resistant to all known antituberculous agents [START_REF] Udwadia | India's multidrug-resistant tuberculosis crisis[END_REF]. At Hinduja Hospital (Mumbai, India), we received ethics committee approval to proceed with a trial involving addition of TDZ on a 'compassionate use basis'. Eight patients with XDR-TB who remained persistently sputum smear-and culture-positive despite 1 year of second-line drugs have been enrolled to receive TDZ in escalating doses. Baseline cardiac status and regular monitoring for QTc prolongation were and will be performed throughout the duration of the study. It is hoped that this study will pave the way for larger collaborative studies with TDZ in In this era of MDR-and XDR-TB, we need a smart drug development programme for clinical evaluation of potential antituberculous drugs in parallel [START_REF] Mitnick | Randomized trials to optimize treatment of multidrug-resistant tuberculosis[END_REF]. In the specific case of TDZ, the approach can be tailor-made: since there is such a long clinical experience with this drug, a combined phase I and II study in normal sensitive TB patients with assessment of early bactericidal activity at increasing dosages can be designed. A small phase II study looking at safety and tolerability as well as evaluation of serial sputum colony counts [START_REF] Mitchison | Clinical development of anti-tuberculosis drugs[END_REF] for TDZ in combination with a standard regimen in normal sensitive TB can follow. Consecutively, there should be a 'smart' trial design in MDR-or XDR-TB patients in two groups: a control group receiving best care and a trial group receiving best care plus TDZ (Tibotec model presented at the 46th Interscience Conference on Antimicrobial Agents and Chemotherapy, 2008). There are several sites in Africa and Asia that have developed or are in the process of developing clinical research capacity to make these kinds of approaches feasible.

In conclusion, there is a pressing need to evaluate drugs such as TDZ in clinical trials for the therapy of XDR-TB as described above. To conduct these global A c c e p t e d M a n u s c r i p t trials there is the need to create international laboratories to serve as centres for rapid identification of XDR-TB patients and that can accurately characterise the resistance spectrum. The situation is urgent; we must work fast and secure the support needed to perform international standard regulatory studies that will influence TB programmes. Thus, we must create the political will within each country, as support from the respective governments and their health agencies is vital for any success. It is the responsibility of the whole healthcare community to support endeavours to develop new treatments for XDR-TB urgently. Please join us and the supporters of global trials for therapy of XDR-TB identified in this article.

  ) has been shown to have in vitro activity against multidrugresistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis, to promote the killing of intracellular MDR and XDR strains and to cure the mouse of antibiotic-susceptible and -resistant pulmonary tuberculosis (TB) infections. Recently, TDZ was used to cure 10 of 12 XDR-TB patients in Buenos Aires, Argentina. At the time of writing, it is being used for the therapy of non-antibiotic-responsive terminal XDR-TB patients in Mumbai, India, on the basis of compassionate therapy and although it is too early to determine a cure, the patients have improved appetite, weight gain, are afebrile and free of night sweats, and their radiological picture shows great improvement. Because XDR-TB is essentially a terminal disease in many areas of the world and no new effective agents have yet to yield successful clinical trials, global clinical trials for the therapy of XDR-TB are urgently required.

  s c r i p t patients with XDR-TB who have exhausted all available drug options. At the time of writing, all the patients have tolerated daily doses of 75 mg TDZ during the first 6 weeks of therapy with encouraging results in terms of clinical well-being (cessation of coughing, afebrile, regaining loss weight, etc.). Sputum culture reports are awaited.

Funding

None.

Competing interests

None declared.

Ethical approval

Not required. Tegos, Massachusetts, USA (GTEGOS@PARTNERS.ORG); Jakko van Ingen, Bilthoven, The Netherlands (Jakko.van.Ingen@rivm.nl); Andras Varga, IMP, HU, Berlin, Germany (andrasv@aol.com); Jordi Vila, Barcelona, Spain (jvila@ub.edu); Miguel Viveiros, IHMT, Lisbon, Portugal; Rolf Walter, BNITM, Hamburg, Germany (rolfdwalter@yahoo.de); and Kanglin Wan, CDC, Beijing, China (wankanlgin@icdc.cn).


Andrew P Desbois

Curtis G Gemmell

Peter J Coote

In vivo efficacy of the antimicrobial peptide ranalexin in combination with the endopeptidase lysostaphin against wound and systemic meticillin-resistant Staphylococcus aureus (MRSA) infections

Keywords:

resistant Staphylococcus aureus (MRSA) as these strains are often resistant to multiple conventional antibiotics. Earlier studies showed that ranalexin, an antimicrobial peptide (AMP), in combination with lysostaphin, an antistaphylococcal endopeptidase, synergistically inhibits the growth of MRSA, meaning that it deserved consideration as a new anti-S. aureus therapy. Using haemolysis and Vero cell viability assays, ranalexin with lysostaphin is proven to be non-toxic at antibacterial concentrations. In human serum, ranalexin with lysostaphin is significantly more effective against MRSA than treatment with either component alone. In a rabbit model of wound infection, ranalexin with lysostaphin reduced MRSA in the wound by ca. 3.5 log 10 colony-forming units (CFU) compared with the untreated control. The combination is significantly more effective than treatment with ranalexin or lysostaphin alone. In a mouse model of systemic infection, ranalexin with lysostaphin reduced MRSA kidney burden by ca. 1 log 10 CFU/g compared with untreated controls or treatment with ranalexin or lysostaphin alone.

Importantly, the combination is synergistically bactericidal against various S. aureus isolates in vitro, including those with reduced susceptibility to lysostaphin or vancomycin. Ranalexin and lysostaphin could be incorporated in wound dressings for the prevention and treatment of topical S. aureus infections. That AMPs can enhance the antibacterial effectiveness of lysostaphin in vivo highlights a new avenue of research in the fight against drug-resistant staphylococci.

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

Introduction

Staphylococcus aureus is a serious opportunistic pathogen that can infect open wounds such as burns, ulcers and sites of surgery and can cause potentially lethal systemic infections [START_REF] Gemmell | Guidelines for the prophylaxis and treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections in the UK[END_REF]. Meticillin-resistant S. aureus (MRSA) strains are a particular problem as infections caused by these pathogens are more difficult and expensive to treat [START_REF] Tai | Methicillin-resistant Staphylococcus aureus in orthopaedic surgery[END_REF][START_REF] Gould | Costs of hospital-acquired methicillin-resistant Staphylococcus aureus (MRSA) and its control[END_REF]. Moreover, MRSA strains can be resistant to multiple antibiotics, meaning that effective treatment can be unreliable, especially with the emergence of strains with reduced susceptibility to the 'antibiotic of last resort', vancomycin [START_REF] Boyle-Vavra | Development of vancomycin and lysostaphin resistance in a methicillin-resistant Staphylococcus aureus isolate[END_REF]. Thus, new treatments for topical and systemic MRSA infections are urgently required.

Antimicrobial peptides (AMPs) are considered to have potential as new therapeutic agents for treating drug-resistant bacterial infections [START_REF] Hancock | Peptide antibiotics[END_REF][START_REF] Ghiselli | Therapeutic efficacy of the polymyxin-like peptide ranalexin in an experimental model of endotoxemia[END_REF][START_REF] Andrès | Cationic peptides: update of clinical development[END_REF].

Concomitantly, the antistaphylococcal endopeptidase lysostaphin has attracted attention in the treatment of drug-resistant S. aureus infections [START_REF] Patron | Lysostaphin treatment of experimental aortic valve endocarditis caused by a Staphylococcus aureus isolate with reduced susceptibility to vancomycin[END_REF][START_REF] Kokai-Kun | Lysostaphin as a treatment for systemic Staphylococcus aureus infection in a mouse model[END_REF].

Recently, it has been shown that combinations of AMPs with lysostaphin achieve enhanced or synergistic antibacterial effects against S. aureus and MRSA compared with the compounds individually [10,11]. One such synergistic combination consists of lysostaphin with ranalexin, a cationic AMP originally isolated from the North American bullfrog, Rana catesbeiana [START_REF] Clark | A novel antimicrobial peptide from bullfrog (Rana catesbeiana) skin, structurally related to the bacterial antibiotic, polymyxin[END_REF]. This bactericidal combination kills bacteria within 5 min, is effective in low pH and high salt conditions and, importantly, is effective on human skin [11].

Moreover, individually ranalexin and lysostaphin are efficacious and non-toxic in animal models of bacterial infection [START_REF] Ghiselli | Therapeutic efficacy of the polymyxin-like peptide ranalexin in an experimental model of endotoxemia[END_REF][START_REF] Giacometti | Efficacy of polycationic peptides in preventing vascular graft infection due to Staphylococcus epidermidis[END_REF][START_REF] Giacometti | Polycationic peptides as prophylactic agents against methicillinsusceptible or methicillin-resistant Staphylococcus epidermidis vascular graft infection[END_REF]. These earlier observations A c c e p t e d M a n u s c r i p t suggested that ranalexin in combination with lysostaphin warranted further assessment for its ability to treat MRSA infections in vivo.

The present study was aimed to assess whether the combination of ranalexin with lysostaphin was effective in the treatment of wound and systemic MRSA infections in animal models and whether this combination was more effective compared with each component individually.

Materials and methods

Reagents, culture media and microorganisms

High-performance liquid chromatography (HPLC)-grade reagents (or the highest grade available) and culture media were purchased from Sigma Aldrich Ltd. (Poole, UK). Ranalexin (>95% purity) and recombinant lysostaphin (>93% purity) were sourced as previously [11]. Stocks and all other solutions were made with ultrapure deionised water unless stated.

Efficacy in human serum

Briefly, 48 L of MRSA252 cell suspension [1  10 6 colony-forming units (CFU)/mL, exponential phase] made up in heat-inactivated human serum (Lonza, Wokingham, UK) was added to each well of a 96-well plate. The bacterial inoculum had been cultured in a shaking incubator (37 C, 220 rpm) from a single colony in tryptone soya broth (TSB). Then, 2 L of stock solutions was added to experimental wells to give final well concentrations of 25 mg/L ranalexin alone, 0.125 mg/L lysostaphin alone or these compounds together at these concentrations; water only was added to control wells. Each treatment was performed in quadruplicate. Following incubation (ca. 25 C, 35 min), viable bacteria were determined for each well by serial dilution in phosphate-buffered saline (PBS) [for 1 L: 8 g NaCl, 0.2 g KCl, 1.15 g Na 2 HPO 4 , 0.2 g KH 2 PO 4 (pH 7.3)] and plating on tryptone soya agar (TSA).

Plates were incubated (37 C, 24 h) and colony counts were performed. The number of colonies recovered from replicate wells for each treatment were transformed by log 10 and then averaged to give a mean value.

Haemolysis assay

To each well of a 96-well plate was added 96 L of 2% defibrinated horse blood (ca. 1.3  10 8 erythrocytes/mL) (Oxoid, Basingstoke, UK) in PBS and 4 L of various stock solutions to give final well concentrations of 10-100 mg/L A c c e p t e d M a n u s c r i p t ranalexin, 0.1-5 mg/L lysostaphin or these compounds in combination; water only was added to negative control wells. For positive controls, erythrocytes were suspended in 1% sodium dodecyl sulphate in water, which causes 100% haemolysis. The plate was incubated (37 C, 4 h, shaker speed 2) on a microplate spectrophotometer (PowerWave XS with KC4 software; Bio-Tek Instruments Inc., Winooski, VT) with A 570 readings collected every 2 min. A reduction in A 570 indicated haemolysis.

Mammalian cell toxicity assay

Mammalian cell toxicity was assessed using the neutral red assay modified from Babich and Borenfreund [START_REF] Babich | Cytotoxicity of T-2 toxin and its metabolites determined with the neutral red cell viability assay[END_REF]. Vero cells, routinely cultured in 75 cm 2 flasks (5% CO 2 , 95% humidity, 37 C, ca. 72 h) in 5 mL of RPMI 1640 medium supplemented with 10% bovine calf serum (Cambrex Corp., East Rutherford, NJ), penicillin (50 000 U/L) and streptomycin (50 mg/L), were exposed to trypsin and harvested with fresh medium. Then, 40 000 cells (200 L) was added to each well of a 96-well plate and the plate was incubated for 24 h to allow cell monolayers to develop. The medium was then removed and replaced with 196 L of fresh medium and 4 L of various stock solutions to give the same concentrations of ranalexin and lysostaphin as described in Section 2.3. The plate was incubated for 48 h. The medium was removed, the cells were washed with 200 L of PBS and then 100 L of fresh medium supplemented with neutral red (100 mg/L) was added to each well. The plate was incubated (90 min), the medium was removed and the cells were washed with PBS before the addition of 200 L of acidified isopropanol (0.33% HCl) to A c c e p t e d M a n u s c r i p t each well. A 540 was determined for each well, with a reduction in A 540 indicating reduced cell viability.

Spectrum of activity

To determine the spectrum of activity, 96 L of bacterial suspensions in TSB (1  10 6 CFU/mL, exponential phase) and 4 L of stock solutions was added to wells on a 96-well plate to give various final well concentrations of ranalexin alone, lysostaphin alone or ranalexin with lysostaphin; to control wells was added water only. Each treatment was performed in quadruplicate. Bacterial inocula were prepared as described in Section 2.2 in either TSB, TSB supplemented with 2 mg/L vancomycin for the VISA isolates or TSB supplemented with 10 mg/L erythromycin for the lysostaphin-resistant mutants (as erythromycin is the selectable marker for the these transposon mutants from the Phoenix library [START_REF] Gründling | Staphylococcus aureus mutants with increased lysostaphin resistance[END_REF]). The 96-well plate was incubated (37 C, 24 h, 1000 rpm) on a microplate thermoshaker (PHMP; Grant Instruments Ltd., Shepreth, UK). Following incubation, viable bacteria were determined for each well by serial dilution in PBS and plating on TSA as described in Section 2.2.

Bactericidal synergy is defined as a >2 log 10 CFU/mL reduction by the combination treatment compared with its most active constituent, providing that the number of viable bacteria recovered from the combination treatment is >2 log 10 CFU/mL lower than the starting inoculum [START_REF] White | Comparison of three different in vitro methods of detecting synergy: time-kill, checkerboard, and E test[END_REF]. Furthermore, at least one of the components in the combination must not affect the viability of the test organism when used alone.

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

Rabbit model of MRSA wound infection

Animal experimentation was performed by Huntingdon Life Sciences and adhered to their in-house ethics policies. The following method is modified from Morton and Malone [START_REF] Morton | Evaluation of vulnerary activity by an open wound procedure in rats[END_REF]. New Zealand White rabbits (1.71-3.31 kg, 10-15 weeks old, outbred) were housed in polypropylene or metal cages with grid floors operating a 12 h:12 h light:dark cycle (18 C, 55% relative humidity) and given access to food and water ad libitum. Each rabbit was anaesthetised with isoflurane and a 3 cm  3 cm area of the dorsal thoracic region was shaved and disinfected with 5% chlorhexidine digluconate. The area was swabbed and plated on mannitol salt agar (MSA) but no contaminants were ever recovered. A circular wound (ca. 1.5 cm diameter) was made by excision (including the panniculus carnosus and adherent tissues) and inoculated with ca. 1  10 9 CFU of MRSA4 in 0.2 mL of PBS (prepared from surface growth on TSA). The wound was covered with a sterile Hilltop chamber (gauze moistened with saline), which was secured with a dressing and further covered to prevent exposure to the air.

To recover from the anaesthetic, each animal received subcutaneous injections of 0.05 mg/kg buprenorphine immediately after wounding and at 8, 16 and 24 h thereafter. Animals were assigned to five treatment groups (two male and two female mice for each experimental group and one male and one female mice for the two control groups): (i) PBS (untreated control); (ii) ranalexin 0.225 mg; (iii) lysostaphin 0.0045 mg; (iv) ranalexin 0.225 mg + lysostaphin 0.0045 mg; and (v) vancomycin 6 mg (positive control). These doses were administered at 24, 48 and 72 h post inoculation in 0.3 mL of PBS A c c e p t e d M a n u s c r i p t by moistening the gauze of fresh Hilltop chambers. Every 24 h, gauzes from the Hilltop chambers were removed from the wounds and were assessed for MRSA4 burden by vortexing the whole gauze in PBS, serially diluting the liquid and plating on MSA supplemented with enrofloxacin (MSA-E) to select for MRSA4. At 96 h post inoculation, the rabbits were humanely sacrificed with pentobarbital and the wound area was carefully excised, homogenised in PBS, diluted and plated on MSA-E to assess MRSA4 burden. Group sizes for the untreated control and the vancomycin positive control were smaller than the experimental groups owing to the high cost of this experimentation.

Furthermore, the key aim in this experiment was to determine whether there was a beneficial effect of combining ranalexin with lysostaphin compared with the individual components alone. Mice were inspected for adverse clinical signs at inoculation every 2 h for the first 6 h and then at 6 h intervals. Body weights were recorded prior to inoculation and then at 20, 44 and 68 h. At 74 h post inoculation, animals were sacrificed and MRSA4 kidney burden was assessed by excising the kidneys. Kidneys were weighed, homogenised in PBS (in pairs), serially diluted and plated on MSA. Plates were incubated (37 C, 24 h) and colony counts were performed and converted to CFU/g of tissue.

Murine model of systemic MRSA infection

Statistical analyses

All statistical tests were performed using SPSS v15.0 for Windows (SPSS Inc., Chicago, IL). For the human serum and in vivo data, log 10 CFU reductions (per mL, per g or in total) were compared between groups by Mann-Whitney U-test (one-sided) employing Holm's correction to P-values for multiple comparisons [START_REF] Holm | A simple sequentially rejective multiple test procedure[END_REF]. For the haemolysis and Vero cell experiments, Kruskal-Wallis tests were performed on the raw data to compare the combination groups and the 50 mg/L ranalexin treatment and where significant (i.e. heterogeneity between groups detected) this was followed by subsequent Mann-Whitney U-tests. In all cases, P  0.05 was considered statistically significant.

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

Results

Efficacy in human serum

To assess the usefulness of the combination for subsequent testing in animal models, an evaluation of efficacy was performed in human serum. Untreated controls showed no change in viable MRSA252 during 35 min of incubation [0.00 ± 0.03 log 10 CFU/mL (mean ± 1 standard error)]. Ranalexin at 25 mg/L had little effect on bacterial abundance (increase of 0.10 ± 0.03 log 10 CFU/mL), whilst lysostaphin at 0.125 mg/L reduced viable bacteria by 1.25 ± 0.37 log 10 CFU/mL. In combination, ranalexin with lysostaphin reduced viable MRSA252 by 3.65 ± 0.14 log 10 CFU/mL, which was a significantly greater reduction compared with treatment with ranalexin alone (P < 0.05) or lysostaphin alone (P < 0.05). Thus, even in human serum, which contains components that can inhibit the action of antibiotics [START_REF] Ciornei | Antimicrobial and chemoattractant activity, lipopolysaccharide neutralization, cytotoxicity, and inhibition by serum of analogs of human cathelicidin LL-37[END_REF], the combination is antibacterial and has enhanced efficacy compared with either ranalexin or lysostaphin alone.

In vitro toxicity

Ranalexin alone caused haemolysis and showed signs of toxicity to Vero cells at 50 mg/L, but lysostaphin alone caused no haemolysis and had no effect on Vero cell viability. In both assays, ranalexin in combination with lysostaphin was no more toxic than the ranalexin component alone (Vero toxicity, H 4,30 = 3.864; P > 0.05; haemolysis, H 4,30 = 10.591; P < 0.05, but Mann-Whitney Utests confirmed that each combination caused no greater haemolysis compared with ranalexin alone) (Table 1). Reassuringly, the concentrations of A c c e p t e d M a n u s c r i p t the combination that were toxic to erythrocytes and Vero cells were in excess of those required for effective antibacterial activity in human serum.

Spectrum of activity

The synergistic antibacterial efficacy of ranalexin with lysostaphin was assessed against various strains of S. aureus, including lysostaphin-resistant mutants, clinically-relevant VISA isolates and MRSA4 (used in animal infection models) as well as a strain of S. epidermidis. In combination, ranalexin with lysostaphin synergistically reduced the viability of each S. aureus isolate and the S. epidermidis strain (Table 2). The concentrations of ranalexin and lysostaphin selected for these experiments were dependent on the isolate under investigation, as the susceptibility of each isolate differed with respect to each component. In addition, while some of these concentrations are high enough to be toxic in vitro, they were selected to best highlight the synergistic interaction (lower, non-toxic concentrations are bactericidal but do not show the synergy as distinctly).

Rabbit model of MRSA wound infection

Wound dressings that had been treated with PBS, ranalexin alone, lysostaphin alone, ranalexin with lysostaphin, or vancomycin were applied to infected wounds for 24 h and then replaced with freshly treated dressings. Upon removal from the wound, the abundance of MRSA4 was assessed for each dressing to provide an indication of infection progression and bacterial survival on the dressing. During the experiment, mean viable MRSA4 recovered from A c c e p t e d M a n u s c r i p t the dressings containing the combination treatment reduced from ca. 7.5 log 10 CFU at 48 h post inoculation to ca. 3 log 10 CFU at 96 h (Fig. 1). At 96 h post inoculation, the dressings containing the combination contained fewer viable MRSA4 than those containing lysostaphin alone by ca. 2 log 10 CFU (U = 3; n 1 = n 2 = 4; P > 0.05) and ranalexin alone by ca. 3.5 log 10 CFU (U = 1; n 1 = n 2 = 4; P < 0.05) (Fig. 1). These data confirm that the combination of ranalexin with lysostaphin is effective at reducing the viability of MRSA4 when soaked into a gauze dressing.

The combination treatment of ranalexin with lysostaphin reduced median viable MRSA4 recovered from infected wounds by ca. 3.5 log 10 CFU compared with the untreated control and ranalexin alone (U = 0; n 1 = n 2 = 4; P < 0.05) groups and by ca. 2 log 10 CFU compared with treatment with lysostaphin alone (U = 1; n 1 = n 2 = 4; P < 0.05) (Fig. 2). The combination was more efficacious than vancomycin treatment as the combination reduced viable MRSA4 by a further ca. 2.5 log 10 CFU (Fig. 2). The combination treatment significantly reduced viable MRSA4 compared with lysostaphin alone (P < 0.05) and ranalexin alone (P < 0.05), confirming that when soaked into a dressing this combination acts against MRSA in an infected wound with enhanced efficacy compared with treatment with each component of the combination alone.

Murine model of systemic MRSA infection

The combination treatment reduced median viable MRSA4 recovered from the animals' kidneys by ca. 1 log 10 CFU/g compared with the untreated control or A c c e p t e d M a n u s c r i p t treatment with ranalexin alone or lysostaphin alone (Fig. 3). However, the combination treatment was no more effective statistically than treatment with lysostaphin alone (U = 22; n 1 = n 2 = 8; P > 0.05). In the positive control group (vancomycin 50 mg/kg), median viable MRSA4 recovered from the animals' kidneys reduced by ca. 4.5 log 10 CFU/g, whilst no MRSA4 was recovered from four of eight animals, indicating complete clearance of infection in these cases.

Body weights reduced each day in each treatment group after inoculation (data not shown), with the greatest change in the ranalexin treatment group (-16.3% at 68 h post inoculation). Although the reduction in body weight in the combination treatment group (-10.5 %) was less than the reduction in the lysostaphin only treatment group (-12.3 %), this difference was not significant (U = 23.5; n 1 = n 2 = 8; P > 0.05). Body weights changed in the PBS and vancomycin treatment groups by -14.3 % and -3.3 %, respectively. In general, few adverse clinical signs were observed, but towards the end of the study (ca. 68 h) some animals showed slight piloerection (3/8 in the PBStreated group, 7/8 in the ranalexin-treated group, 2/8 in the lysostaphin-treated group and 3/8 in the combination-treated group). In the combination treatment group, one animal developed a slightly bruised tail at 32 h. The animals' tails in the ranalexin treatment group showed slight bruising (5/8), slight swelling (1/8) and reddening (3/8), but these clinical signs were always mild.

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

Discussion

In combination, ranalexin with lysostaphin reduces the burden of MRSA in infected wounds and treatment dressings to a greater extent than treatment with each component of the combination alone. This is the first demonstration that an AMP administered in combination with lysostaphin can achieve enhanced antibacterial activity in vivo compared with either component alone.

The combination of ranalexin with lysostaphin synergistically kills MSSA, MRSA, VISA and lysostaphin-resistant strains of S. aureus and a clinical isolate of S. epidermidis. This combination is effective in human serum and reduces the bacterial burden in a mouse model of systemic MRSA infection.

In contrast to a previous report [START_REF] Clark | A novel antimicrobial peptide from bullfrog (Rana catesbeiana) skin, structurally related to the bacterial antibiotic, polymyxin[END_REF], ranalexin and combination treatments containing ranalexin were found to be toxic to erythrocytes and Vero cells in vitro at concentrations close to those needed for potent bactericidal activity in vitro. However, perhaps more importantly, only mild signs of toxicity were detected in the mouse systemic model, whilst there was no evidence for toxicity in the rabbit wound model. Moreover, other studies have shown ranalexin to be non-toxic at efficacious antibacterial concentrations in vivo [START_REF] Giacometti | Efficacy of polycationic peptides in preventing vascular graft infection due to Staphylococcus epidermidis[END_REF][START_REF] Giacometti | Polycationic peptides as prophylactic agents against methicillinsusceptible or methicillin-resistant Staphylococcus epidermidis vascular graft infection[END_REF], suggesting that the highly effective combination of ranalexin with lysostaphin could still have potential medical applications.

Topically applied agents are important in the prevention and management of wound infections. Compared with systemic antibiotics, topical therapies benefit from lower toxicity and higher local concentrations of the antibacterial agent, especially where the blood supply to the wound is poor [START_REF] Fletcher | Best practice-choosing an appropriate antibacterial dressing[END_REF]. Presently, A c c e p t e d M a n u s c r i p t all available antibacterial dressings contain broad-spectrum agents, but such agents and non-specific biocides can actually impair wound healing processes [START_REF] Peter | The effect of wound ointments on tissue microcirculation and leucocyte behaviour[END_REF][START_REF] Vick | Effect of Dakin's solution on components of a dermal equivalent[END_REF]. Moreover, broad-spectrum agents may kill microorganisms that live harmlessly on the skin or in the wound and provide niche protection against opportunistic pathogens [START_REF] Cooper | A review of the evidence for the use of topical antimicrobial agents in wound care[END_REF]. A dressing containing ranalexin with lysostaphin could be used as a prophylactic to reduce the chances of contracting an infection, as its narrow spectrum of activity should not affect the normal skin flora but would prevent staphylococci from colonising the dressing itself or the wound area. Alternatively, the dressing could be applied once an infection has become established. Also, wound exudate would not be expected to interfere with the combination's efficacy, as it remains effective in human serum and wound exudate contains similar components to serum [START_REF] White | Modern exudate management: a review of wound treatments[END_REF].

Combination therapies greatly reduce the opportunity for resistant strains to be selected provided that two mechanisms of action are used by the drugs in question [START_REF] Zhao | Restricting the selection of antibiotic-resistant mutants: a general strategy derived from fluoroquinolone studies[END_REF]. Furthermore, the narrow spectrum of the ranalexin with lysostaphin treatment reduces the opportunity for resistance to emerge in the normal bacterial flora, which is a mechanism recognised for the emergence and spread of resistance to pathogens [START_REF] Coates | The future challenges facing the development of new antimicrobial drugs[END_REF]. Importantly, the present study shows that in combination ranalexin with lysostaphin is synergistically bactericidal against S. aureus strains with reduced susceptibility to lysostaphin, which is often characterised by mutation of the fem or lyrA genes [START_REF] Gründling | Staphylococcus aureus mutants with increased lysostaphin resistance[END_REF][START_REF] Climo | Mechanism and suppression of lysostaphin resistance in oxacillin-resistant Staphylococcus aureus[END_REF]. Staphylococcus aureus mutants with reduced susceptibility to ranalexin have not been found [START_REF] Giacometti | In-vitro activity and killing effect of polycationic peptides on methicillin-resistant Staphylococcus aureus and interactions with clinically used antibiotics[END_REF] and are thought to be rare due to its nonspecific mode of action. The combination of ranalexin with lysostaphin was A c c e p t e d M a n u s c r i p t effective and synergistic against VISA isolates but an assessment is required for its effectiveness against further important strains, including communityacquired MRSA (CA-MRSA).

Additional medications are urgently required for MRSA owing to the emergence of resistance to the 'last resort' antibiotic, vancomycin [START_REF] Boyle-Vavra | Development of vancomycin and lysostaphin resistance in a methicillin-resistant Staphylococcus aureus isolate[END_REF], as well as the newly introduced agents daptomycin [START_REF] Marty | Emergence of a clinical daptomycin-resistant Staphylococcus aureus isolate during treatment of methicillin-resistant Staphylococcus aureus bacteremia and osteomyelitis[END_REF], linezolid [START_REF] Tsiodras | Linezolid resistance in a clinical isolate of Staphylococcus aureus[END_REF], quinupristin/dalfopristin [START_REF] Werner | Methicillin-resistant, quinupristin-dalfopristin-resistant Staphylococcus aureus with reduced sensitivity to glycopeptides[END_REF] and teicoplanin [START_REF] Cepeda | Teicoplanin resistance in methicillin-resistant Staphylococcus aureus in an intensive care unit[END_REF]. Concerns have been raised regarding the safety of lysostaphin with respect to inducing adverse immunological reactions; however, these responses seem unapparent even after prolonged or systemic application [START_REF] Quickel | Efficacy and safety of topical lysostaphin treatment of persistent nasal carriage of Staphylococcus aureus[END_REF][START_REF] Harrison | Antigenic response to topically applied proteins[END_REF][START_REF] Daley | Lysostaphin: immunogenicity of locally administered recombinant protein used in mastitis therapy[END_REF][START_REF] Climo | Lysostaphin treatment of experimental methicillin-resistant Staphylococcus aureus aortic valve endocarditis[END_REF][START_REF] Dajcs | Immunity to lysostaphin and its therapeutic value for ocular MRSA infections in the rabbit[END_REF]. Hence, considering its efficacy, the combination of ranalexin with lysostaphin may be useful in the Prior to application to the wound, dressings were treated with phosphatebuffered saline only (), 0.225 mg ranalexin (○), 0.0045 mg lysostaphin (■), 0.225 mg ranalexin + 0.0045 mg lysostaphin ( ), or 6 mg vancomycin (▲).

Freshly treated dressings were applied every 24 h. The limit of detection was 100 CFU/mL. Arithmetic means plotted; n = 4 (except for control and vancomycin groups where n = 2); error bars indicate +1 standard error. CFU, colony-forming units. a Note that a negative value in the table indicates that haemolysis was less than the control and that Vero cell viability was greater than the control. In both assays, n = 6; ±1 standard error.

Edited Table 1 A c c e p t e d M a n u s c r i p t a The detection limit was 2 log 10 CFU/mL. n = 4; data given as mean (standard deviation)

b Parent strain of ANG133 and ANG144.

c Parent strain of ANG365 and ANG366 [START_REF] Ciornei | Antimicrobial and chemoattractant activity, lipopolysaccharide neutralization, cytotoxicity, and inhibition by serum of analogs of human cathelicidin LL-37[END_REF].

d Exposure was 30 h for Mu50 as this is a slow-growing strain.

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 ABSTRACT New treatments are urgently required for infections caused by meticillin-

  Solutions and media were autoclaved at 121 C for 15 min or filter-sterilised (polyethersulfone 0.22 m; Millipore, Watford, UK). Stocks of the following S. aureus isolates were kind gifts: meticillin-resistant strain MRSA252 and meticillin-susceptible S. aureus (MSSA) strain MSSA476 (from Prof. Mark Enright, Imperial College, London, UK); BB270, Newman and four lysostaphin-resistant mutants (from Dr Angelika Gründling, Imperial College London, UK); six isolates with reduced susceptibility to vancomycin [vancomycin-intermediate S. aureus (VISA) isolates] (from Dr Sue Lang, Glasgow Caledonian University, UK); RN4220 and SH1000 (from Prof. Simon

A

  c c e p t e d M a n u s c r i p t Foster, University of Sheffield, UK); and MRSA4 (Huntingdon Life Sciences, Alconbury, UK). Staphylococcus epidermidis was an in-house clinical isolate.

Female

  ICR CD-1 mice (22-26 g, 4-6 weeks old) were housed in vented polycarbonate cages operating a 12 h:12 h light:dark cycle (21 C, 55% relative humidity) and given access to food and water ad libitum. Each mouse was inoculated intravenously via the tail vein with ca. 1.7  10 7 CFU of MRSA4 (prepared in 0.2 mL of 2% mucin in beef broth from surface growth on TSA). Animals were randomly assigned to five treatment groups (eight mice per group): (i) PBS; (ii) ranalexin 12 mg/kg; (iii) lysostaphin 0.1 mg/kg; (iv) ranalexin 12 mg/kg + lysostaphin 0.1 mg/kg; and (v) vancomycin 50 mg/kg. Doses (all in PBS) were administered intravenously at 2, 26 and 50 h post inoculation (dose volume 10 mL/kg) with the exception of vancomycin, which was administered by subcutaneous injection into the skin around the neck.

A

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

  prevention and treatment of drug-resistant-S. aureus infections, especially for topical application in a gel [11] or incorporated into a wound dressing. That AMPs can enhance the antibacterial effectiveness of lysostaphin in vivo highlights a new avenue of research in the fight against drug-resistant staphylococci. 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 [10] Graham S, Coote PJ. Potent, synergistic inhibition of Staphylococcus aureus upon exposure to a combination of the endopeptidase lysostaphin and the cationic peptide ranalexin. J Antimicrob Chemother 2007;59:759-62. [11] Desbois AP, Lang S, Gemmell CG, Coote PJ. Surface disinfection properties of the combination of an antimicrobial peptide, ranalexin, with an endopeptidase, lysostaphin, against methicillin-resistant Staphylococcus aureus (MRSA). J Appl Microbiol 2010;108:723-30.

Fig. 1 .

 1 Fig. 1. Recovery of viable meticillin-resistant Staphylococcus aureus strain

Fig. 2 .

 2 Fig. 2. Recovery of viable meticillin-resistant Staphylococcus aureus strain

Fig. 3 .

 3 Fig. 3. Recovery of viable meticillin-resistant Staphylococcus aureus strain

Table 1

 1 Toxicity of ranalexin and lysostaphin alone and in combination compared with

	untreated controls as assessed by (i) haemolysis of equine erythrocytes
	during 4 h incubation and (ii) reduction in viability of Vero cells during 48 h
	incubation
	Concentration of	Concentration of	Haemolysis	Reduction in cell
	lysostaphin (mg/L)	ranalexin (mg/L)	(%)	viability (%) a
	Ranalexin alone
	0	10	-3.54 ± 0.90 3.97 ± 2.79
	0	20	-0.07 ± 0.58 -3.15 ± 1.60
	0	30	-0.18 ± 0.48 1.51 ± 1.38
	0	40	0.62 ± 0.55	8.87 ± 2.28
	0	50	20.0 ± 10.9	28.9 ± 9.56
	0	100	44.5 ± 5.09	75.6 ± 4.23
	Lysostaphin alone
	0.1	0	-3.35 ± 0.77 3.04 ± 1.94
	0. 5	0	-2.89 ± 0.78 -1.08 ± 1.66
	1	0	-1.56 ± 0.40 2.71 ± 1.44
	5	0	-3.32 ± 0.53 -3.13 ± 1.24
	In combination
	0.1	50	26.0 ± 11.7	43.7 ± 2.83
	0. 5	50	4.04 ± 0.56	41.0 ± 5.66
	1	50	-0.28 ± 0.70 36.9 ± 1.53
	5	50	6.86 ± 2.44	43.6 ± 8.19

Table 2

 2 Effect on the viability of Staphylococcus aureus isolates and an isolate of Staphylococcus epidermidis during 24 h exposure to ranalexin, lysostaphin or ranalexin with lysostaphin

	Strain	Concentration (mg/L)	Mean log 10 CFU/mL a
		Ranalexin Lysostaphin Inoculum Untreated	Ranalexin	Lysostaphin	Ranalexin with
					control	alone	alone	lysostaphin
	MSSA isolates
	MSSA476	32	0.125	6.42	9.96 (0.06)	9.73 (0.19)	9.80 (0.16)	2.00 (0.00)
				(0.02)
	Newman b	32	0.063	5.93	9.94 (0.06)	9.85 (0.07)	9.82 (0.05)	2.00 (0.00)
				(0.22)
	RN4220	48	0.023	6.20	9.28 (0.06)	9.38 (0.04)	9.39 (0.07)	4.14 (2.51)
				(0.08)
	SH1000	32	0.063	5.94	9.74 (0.07)	9.73 (0.06)	9.53 (0.12)	2.00 (0.00)
				(0.07)
	MRSA isolates

MSSA, meticillin-susceptible S. aureus; MRSA, meticillin-resistant S. aureus; VISA, vancomycin-intermediate S. aureus; CFU, colony-forming units.

Acknowledgment

The authors wish to acknowledge the contribution of Mr Chris Liddle (Huntingdon Life Sciences) for co-ordinating the in vivo experiments.

This work was supported by the Biotechnology and Biological Sciences

Research Council (Follow-on Fund grant number BB/F528106/1) and the Wellcome Trust (Value in People Award).

Funding

Competing interests

The work described here is the subject of an International Patent Application (PCT/GB2007/001157), in which the authors have no financial interest at present.

Ethical approval

Animal experimentation was performed by Huntingdon Life Sciences (Alconbury, UK) and adhered to their in-house ethics policies.

Treatment group

Edited Figure 3


Jana Held

Philipp Zanger

Saadou Issifou

Peter G Kremsner

Benjamin Mordmüller
email: benjamin.mordmueller@uni-tuebingen.de

In vitro activity of tigecycline in Plasmodium falciparum cultureadapted strains and clinical isolates from Gabon

Keywords:

enhanced activity against bacteria and may also be associated with improved antimalarial activity. Using the histidine-rich protein 2 (HRP2) drug sensitivity assay, we determined the geometric mean 50% inhibitory concentrations (IC 50 ) of tigecycline in culture-adapted strains as well as in 23 clinical P. falciparum isolates from Lambaréné, Gabon. These values were compared with other tetracyclines as well as with clindamycin. Assays with 3 days and 6 days of incubation were evaluated to explore the impact of delayed parasite death on drug activity. IC 50 values in clinical isolates after 6 days of incubation were 160.0 nM [95% confidence interval (CI) 114.6-223.4 nM] for tigecycline, 739.4 nM (445.9-1226.1 nM) for doxycycline and 9.2 nM (95% CI 6.6-12.9 nM) for clindamycin. Tigecycline was found to act faster against plasmodia than any of the other antibiotics tested. This study demonstrates the potential of tetracycline derivatives in the development of improved antimalarials.

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

Introduction

Emerging resistance in Plasmodium falciparum against standard treatment regimens has made the quest for new antimalarials a research priority [START_REF] Mordmüller | Malarial parasites vs. antimalarials: never-ending rumble in the jungle[END_REF]. A successful strategy for identifying new therapeutic agents is to exploit drugs that were primarily developed and marketed against bacteria, a commercially more attractive target [2]. A major advantage of this approach is that candidate compounds can be rapidly evaluated in clinical trials once their antiplasmodial activity has been demonstrated. Clindamycin and tetracycline derivatives are notable examples for the great potential of this strategy. Both have become valuable compounds for combination with faster-acting drugs in the current practice of malaria treatment [START_REF] Lell | Clindamycin as an antimalarial drug: review of clinical trials[END_REF][START_REF] Kremsner | Quinine plus clindamycin improves chemotherapy of severe malaria in children[END_REF].

The mechanisms underlying the antimalarial activity of antibiotics have been elucidated only recently. The parasite's apicoplast and mitochondria are of prokaryotic descent and are therefore susceptible to certain antibiotics [START_REF] Goodman | The effects of anti-bacterials on the malaria parasite Plasmodium falciparum[END_REF]. It has been found that apicoplast function is lost in the parasite generations following antibiotic exposure, which explains the phenomenon of parasite death one cycle after exposure, the substantial difference in activity between the first and second cycle, and the slow antimalarial effect of clindamycin and tetracyclines in vivo [START_REF] Goodman | The effects of anti-bacterials on the malaria parasite Plasmodium falciparum[END_REF][START_REF] Dahl | Multiple antibiotics exert delayed effects against the Plasmodium falciparum apicoplast[END_REF][START_REF] Dahl | Tetracyclines specifically target the apicoplast of the malaria parasite Plasmodium falciparum[END_REF].

Glycylcyclines are tetracycline derivatives containing a glycyl amido substitution at position 9. Tigecycline, the 9-t-butylglycylamido derivative of minocycline, is the first marketed compound of this new class of antibiotics. Its increased affinity for the ribosome yields a more potent inhibition of protein synthesis. This is thought to be the A c c e p t e d M a n u s c r i p t basis for tigecycline's improved antibacterial spectrum, including activity against multidrug-resistant species [START_REF] Olson | Functional, biophysical, and structural bases for antibacterial activity of tigecycline[END_REF][START_REF] Bauer | Comparison of tetracycline and tigecycline binding to ribosomes mapped by dimethylsulphate and drug-directed Fe 2+ cleavage of 16S rRNA[END_REF].

We studied the in vitro activity of tigecycline in comparison with clindamycin and doxycycline in clinical isolates of P. falciparum from Lambaréné, Gabon. Additionally, we evaluated the presence of a delayed death phenomenon in plasmodia after treatment with tigecycline using culture-adapted strains. A chloroquine-sensitive (3D7) and a chloroquine-resistant (Dd2) P. falciparum strain were kept in complete culture medium (RPMI 1640, 25 mM HEPES, 2 mM L-glutamine, 50 g/mL gentamicin and 0.5% w/v AlbuMAX TM ) at 37 C, 5% oxygen and 5% haematocrit. Laboratory isolates were synchronised by sorbitol treatment and seeded as ring stages. The histidine-rich protein 2 (HRP2) drug sensitivity assay was performed A c c e p t e d M a n u s c r i p t according to standard procedures [START_REF] Noedl | Simple histidine-rich protein 2 double-site sandwich enzyme-linked immunosorbent assay for use in malaria drug sensitivity testing[END_REF]. However, in those assays incubated for 6 days, 0.16 mL of medium was changed on Days 2 and 4 without replacement of drug.

Methods

Clinical isolates were from patients with mild malaria presenting at the Medical Research Unit of the Albert Schweitzer Hospital in Lambaréné, Gabon, between February 2009 and May 2009. Inclusion criteria were: (i) P. falciparum monoinfection and parasitaemia between 10 3 and 6  10 5 parasites/L blood, assessed by Giemsa-stained thick blood film; (ii) aged 1-18 years; and (iii) no history of intake of antimalarial drugs for at least 1 month. Immediately before standard medical care was initiated, venous blood was collected in lithium heparin tubes. Parasitised blood was processed within 4 h and added to complete culture medium at a haematocrit of 1.5% and an adjusted parasitaemia of 0.05%. Ninety-six-well plates were incubated for 6 days at 37 C in a candle jar.

Samples were only included in the analysis if the drug-free control showed at least twice the HRP2 concentration of the sample with the highest drug concentration. Beyond that, identical methods as for culture-adapted strains were used.

Individual inhibitory concentrations were determined by non-linear regression analysis of log concentration-response curves using the drc-package v0.9.0 of R v2.6.1 (http://www.r-project.org). For each drug assayed, the geometric means of the 50%, 90% and 99% inhibitory concentrations (IC 50 , IC 90 and IC 99 , respectively) were calculated with 95% confidence intervals (CIs). Two-sided paired t-tests were used for

Results and discussion

In vitro activity in culture-adapted strains

The presence of a delayed death phenomenon was assessed by comparing inhibitory concentrations after 3 days and 6 days of incubation. Compared with antiplasmodial activity after 3 days of incubation, activity after 6 days was remarkably higher for all antibiotics. Clindamycin in particular showed very little activity after 3 days but was the most active antibiotic after 6 days of incubation. Among the tetracyclines, tigecycline was the most active compound at both time points, followed by minocycline, doxycycline and tetracycline. Moreover, tigecycline showed a less pronounced delayed death effect than its sister compounds. Comparing the results between strains, 3D7 was significantly less susceptible than Dd2 against clindamycin and tetracycline when incubated for 3 days. However, this difference was not present following prolonged incubation. Tables 1 and2 summarise the geometric mean inhibitory concentrations in culture-adapted strains.

In vitro activity in clinical isolates

Patients aged between 15 months and 18 years of age were included in the study. 3. Tigecycline exhibited approximately five times the activity of doxycycline (IC 50 = 160.0 nM vs. 739.4 nM; P < 0.0001), whilst clindamycin in turn was more than 16-fold more active than tigecycline (9.2 nM vs. 160.0 nM; P < 0.0001). Compared with its activity against culture-adapted strains, the activity of doxycycline in clinical isolates appeared to be reduced (739.4 nM vs. 354.3 nM for 3D7 and 256.0 nM for Dd2). This was due to a decreased sensitivity of certain strains as indicated by an increased range (103.9-12 146.0 nM) and a markedly right-skewed distribution of inhibitory concentrations. Correlation analysis of IC 50 values showed a positive correlation between doxycycline and clindamycin (r = 0.71, P = 0.0002), but no correlation between tigecycline and any of the other drugs.

Geometric mean parasite density at enrolment was 45 174 parasites/L (95% CI 21

Discussion

This study demonstrates a substantial in vitro activity of tigecycline in culture-adapted strains and clinical isolates of P. falciparum. This new compound exhibits approximately five times the activity of doxycycline in field isolates. Moreover, it appears to act faster than any of the other tetracyclines, exhibiting the highest activity at Day 3. The IC 50 values of classic tetracyclines and clindamycin in this study are consistent with those from previous studies using a similar approach [START_REF] Dahl | Multiple antibiotics exert delayed effects against the Plasmodium falciparum apicoplast[END_REF][START_REF] Divo | Oxygen-and time-dependent effects of antibiotics and selected mitochondrial inhibitors on Plasmodium falciparum in culture[END_REF][START_REF] Pradines | In vitro activities of antibiotics against Plasmodium falciparum are inhibited by iron[END_REF][START_REF] Pradines | Antibiotics for prophylaxis of Plasmodium falciparum infections: in vitro activity of doxycycline against Senegalese isolates[END_REF]. This underlines that prolonged incubation is required to assess adequately the antimalarial potential of most antibiotics in vitro.

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

While preparing this manuscript, Starzengruber et al. [START_REF] Starzengruber | Antimalarial activity of tigecycline, a novel glycylcycline antibiotic[END_REF]presented their results from an independent study using clinical isolates from Bangladesh. They found an increased in vitro antimalarial activity of tigecycline compared with classic tetracycline derivatives such as doxycycline, similar to our results. However, owing to their methodologically different approach using 3 days of incubation only, activities for tigecycline and doxycycline were four and six times lower, respectively, compared with our study [START_REF] Starzengruber | Antimalarial activity of tigecycline, a novel glycylcycline antibiotic[END_REF].

Nevertheless, the ratio expressing the activity of tigecycline as a multiple of the IC 50 for doxycycline was similar: 6.1 in the report from Bangladesh and 4.6 in the present study [START_REF] Starzengruber | Antimalarial activity of tigecycline, a novel glycylcycline antibiotic[END_REF]. We could not reproduce the weak correlation between the activities of doxycycline and tigecycline as reported by Starzengruber et al. [START_REF] Starzengruber | Antimalarial activity of tigecycline, a novel glycylcycline antibiotic[END_REF]. This might be due to the wide scatter of doxycycline activities and the heterogeneous parasite population in Central Africa.

Despite its improved activity, clinical use of tigecycline in the treatment of malaria may be limited due to its pharmacokinetic properties. Its rapid distribution into tissues [START_REF] Agwuh | Pharmacokinetics and pharmacodynamics of the tetracyclines including glycylcyclines[END_REF] may expose parasite populations to a prolonged period of subtherapeutic concentrations, thus increasing the risk of resistance. In addition, the need for intravenous administration restricts the use of tigecycline to hospitalised patients. Furthermore, as for any tetracycline derivative, it is not recommended in pregnant women and small children. On the other hand, it might be interesting to investigate this class of compounds with respect to their use in combination with artemisinins for the treatment of severe multiresistant malaria in adults.

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 ABSTRACT Emerging drug resistance in Plasmodium falciparum and its rapid spread in endemic countries have made the quest for new antimalarials a research priority. Tetracycline and its derivatives are well established compounds for combination with faster-acting drugs in the current practice of malaria treatment. Tigecycline is the first marketed derivative of a new class of tetracycline antibiotics. Its altered structure leads to

  Doxycycline hyclate [molecular weight (MW) 512.94] and clindamycin hydrochloride (MW 461.44) were dissolved in H 2 O at a stock solution of 86 mM and 100 mM, respectively; tetracycline hydrochloride (MW 480.90) was dissolved in methanol at 20 mM; and tigecycline (MW 585.65) and minocycline hydrochloride (MW 493.94) were dissolved in dimethyl sulphoxide (DMSO) at 50 mM. Further dilutions were made in complete medium. Drugs were pre-coated on 96-well plates in two-fold (laboratory strains) or three-fold (clinical isolates) dilutions.

  comparison of geometric mean IC 50 values. Correlation of activity between drugs was assessed by Spearman's (non-parametric) test in Stata v10.0 (StataCorp LP, College Station, TX).

A

  c c e p t e d M a n u s c r i p t Informed consent and assent were obtained from the legal representative and the participating child.

A

  c c e p t e d M a n u s c r i p t 750-93 827/L). Of 27 P. falciparum clinical isolates, 22 fulfilled the criteria of successful culture for doxycycline and 23 for both clindamycin and tigecycline. IC 50 values after 6 days of incubation are summarised in Table

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

Table 1

 1 Geometric mean of 50%, 90% and 99% inhibitory concentrations (IC 50 , IC 90 and IC 99 , respectively) (95% confidence interval) of selected antibiotics against the cultureadapted strain 3D7 of Plasmodium falciparum after 3 days and 6 days of incubation a

	Drug	3 days (M) b		6 days (nM) b
		IC 50	IC 90	IC 99	IC 50	IC 90	IC 99
	Tigecycline 2.3 (2.0-	6.9 (5.3-	22.6	220.2	256.7	303.4
		2.8)	8.9)	(13.4-	(174.0-	(203.4-	(240.1-
				38.2)	278.6)	323.8)	383.4)
	Minocycline 5.6 (4.6-	8.8 (6.3-	14.3	263.6	325.3	409.2
		6.8)	12.3)	(8.3-	(253.3-	(289.2-	(327.5-
				24.9)	274.3)	365.8)	511.3)
	Doxycycline 14.6	22.2	35.2	354.3	548.2	882.6
		(11.6-	(16.7-	(20.6-	(297.3-	(425.0-	(584.7-
		18.4)	29.6)	60.2)	422.2)	706.9)	1332.3)
	Tetracycline 134.1	712.9	>1000	359.7	459.0	598.8
		(58.3-	(101.6-		(200.5-	(245.7-	(304.1-
		308.4)	5003.5)		645.1)	857.4)	1179.2)
	Clindamycin 445.4	>1000	>1000	12.2	18.8	30.1
		(184.9-			(10.0-	(15.1-	(21.0-
		1072.5)			15.0)	23.3)	43.2)

a Pooled data from six assays.

b Note that concentrations after 3 days are given in M and after 6 days in nM.

Table 2

 2 Geometric mean of 50%, 90% and 99% inhibitory concentrations (IC 50 , IC 90 and IC 99 , respectively) (95% confidence interval) of selected antibiotics against the cultureadapted strain Dd2 of Plasmodium falciparum after 3 days and 6 days of incubation a

	Drug	3 days (M) b		6 days (nM) b
		IC 50	IC 90	IC 99	IC 50	IC 90	IC 99
	Tigecycline 2.8 (1.9-	12.5	63.3	173.1	225.1	299.7
		4.1)	(6.4-	(10.8-	(107.2-	(131.5-	(158.7-
			24.2)	370.1)	279.6)	385.3)	565.9)
	Minocycline 3.8 (2.5-	12.8
		6.0)

Table 3

 3 Geometric mean of 50%, 90% and 99% inhibitory concentrations (IC 50 , IC 90 and IC 99 , respectively) (95% confidence interval) for tested antibiotics against 23 clinical isolates of Plasmodium falciparum from Gabon after 6 days of incubation

	Drug	IC 50 (nM)	IC 90 (nM)	IC 99 (nM)
	Tigecycline	160.0 (114.6-	367.3 (275.0-490.6)	929.4 (688.1-1255.5)
		223.4)
	Doxycycline	739.4 (445.9-	6884.4 (3959.0-11	66 709.1 (35 442.4-125
	a	1226.1)	971.4)	558.7)
	Clindamycin 9.2 (6.6-12.9)	79.1 (46.2-135.3)	856.3 (373.7-1962.2)

a n = 22 due to one missing value.

Acknowledgments

The authors would like to thank the participating patients and staff of the Albert Schweitzer Hospital (Lambaréné, Gabon) for their participation and support. Many thanks also to Sabine Gabrysch and Dennis Nurjadi for discussing and editing the manuscript.

Funding

This study was supported by the University of Tübingen (Germany) and the Medical Research Unit of the Albert Schweitzer Hospital (Lambaréné, Gabon).

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

In summary, the structural modifications of tigecycline compared with standard tetracyclines appear to confer an increased and accelerated antimalarial activity. Tigecycline itself is unlikely to replace existing drug combination partners on a large scale owing to its pharmacokinetic and safety characteristics. However, its enhanced activity against P. falciparum demonstrates the potential of new tetracycline derivatives in the development of improved antimalarials and calls for further research in this highly promising field.

Competing interests

None declared.

Ethical approval

The investigation was approved by the Ethics Committee of the International Foundation for the Albert Schweitzer Hospital in Lambaréné, Gabon (Ref.: 1/90).


Violeta Rodríguez-Cerrato

Gema Del Prado

Lorena Huelves

Plínio Naves

Vicente Ruiz

Ernesto García

Carmen Ponte

Francisco Soriano

Comparative efficacy of novobiocin and amoxicillin in experimental sepsis caused by -lactam-susceptible and highly resistant pneumococci

Keywords:

Novobiocin MICs for all strains were 0.25-0.5 mg/L. In sepsis, novobiocin and amoxicillin were evaluated at 25, 50, 100 and 200 mg/kg given at 1, 5, 24 and 48 h post bacterial challenge. The most effective regimens in animals infected with the amoxicillin-susceptible strain were 200 mg/kg novobiocin and 25 mg/kg amoxicillin, achieving 100% survival and undetectable organisms in the peritoneum. Among mice infected with amoxicillin-resistant S. pneumoniae, 200 mg/kg novobiocin gave the highest protection (90-100% survivors), followed by 200 mg/kg amoxicillin (60-100%), 100 mg/kg novobiocin (50-87.5%) and 50 mg/kg amoxicillin (14.3-25%). The killing effect of antibiotics in the peritoneum (mean log 10 colony-forming units/mL between treated and control mice) was as follows: 200 mg/kg novobiocin (6.6) > 200 mg/kg amoxicillin (5.6) > 100 mg/kg novobiocin (3.7) > 50 mg/kg amoxicillin (0.7). Total plasma and ultrafiltrate pharmacokinetics of novobiocin (200 mg/kg, single dose) in non-infected mice showed, respectively, half-lives of 151 min and 215 min, area under the concentration-time curves (AUCs) of 945.0 mg h/L and 136.6 mg h/L and maximal concentrations of 147 mg/L and 18 mg/L. Novobiocin may be a promising agent for therapy of highly -lactam-resistant pneumococcal infections.

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

Introduction

The emergence and steady spread of antibiotic-resistant bacterial pathogens are global public health concerns. In particular, the rapid rise of antibiotic resistance among Streptococcus pneumoniae has complicated the therapy of pneumococcal infections both in adults and children [START_REF] Feikin | Mortality from invasive pneumococcal pneumonia in the era of antibiotic resistance, 1995-97[END_REF][START_REF] Macgowan | BSAC Working Parties on Resistance Surveillance. Clinical implications of antimicrobial resistance for therapy[END_REF][START_REF] Tleyjeh | The impact of penicillin resistance on short-term mortality in hospitalized adults with pneumococcal pneumonia: a systematic review and meta-analysis[END_REF][START_REF]World Health Organization. Pneumococcal vaccines[END_REF]. Therapeutic strategies for combating antibiotic-resistant pneumococcal infections are currently being explored, including the possibility of reviving old active drugs.

Based on previous reports showing remarkable in vitro activity of novobiocin against S. pneumoniae [START_REF] Gombert | Susceptibility of multiply antibiotic-resistant pneumococci to the new quinoline antibiotics, nalidixic acid, coumermycin, and novobiocin[END_REF][START_REF] Jones | Should novobiocin be clinically re-evaluated?[END_REF], we have re-evaluated in vitro and in vivo the antipneumococcal activity of this out-of-the-market antibiotic, which was licensed until recently as an antibiotic for human use in many countries such as the USA and in Europe (i.e. Albamycin ® 200 mg capsules for oral administration; Pharmacia-Upjohn).

Novobiocin, an aminocoumarin natural product elaborated by streptomycetes, was widely used as an antibacterial agent in the 1950s and 1960s. Novobiocin is an inhibitor of bacterial DNA gyrase. Like other coumarin products, it binds more tightly to gyrase (as reflected by equilibrium dissociation constants in the range of 10 nM [START_REF] Schmutz | Resistance genes of aminocoumarin producers: two type II topoisomerase genes confer resistance against coumermycin A 1 and clorobiocin[END_REF]) than do typical quinolones, and novobiocin also causes inhibition of ATP binding [START_REF] Sugino | Energy coupling in DNA gyrase and the mechanism of action of novobiocin[END_REF]. Novobiocin was used for the treatment of a variety of infections caused by susceptible organisms, including pneumococcal pneumonia [START_REF] Limson | Novobiocin, a new antibiotic; laboratory and clinical evaluation of thirty patients with bacterial pneumonia[END_REF]. However, several reports published in the 1950s regarding the emergence of resistance and side effects [START_REF] Bridges | Serious reactions to novobiocin[END_REF][START_REF] Cox | Novobiocin jaundice[END_REF][START_REF] Nichols | Novobiocin; a limited bacteriologic and clinical study of its use in forty-five patients[END_REF], along with the development of other antibiotics (i.e. penicillinase-resistant penicillins), likely determined the lack of usage of this otherwise potent and inexpensive antibiotic. The aims of this study were to investigate the in vitro activity, pharmacokinetics and effectiveness of novobiocin therapy using a murine sepsis model caused by amoxicillin-susceptible and -resistant S. pneumoniae strains.

Material and methods

Bacterial strains and antibiotics

Four clinical strains of S. pneumoniae were used in this study, for which the serotype, origin and penicillin minimum inhibitory concentration (MIC) were as follows: strain AR33118, serotype 3, blood, MIC = 0.015 mg/L; strain MJD3693, 19F, cerebrospinal fluid, MIC = 2 mg/L; strain SPC2162, 19A, blood, MIC = 16 mg/L; and strain SPC2552, 23F, respiratory tract, MIC = 32 mg/L). Novobiocin sodium salt (N6160; Sigma-Aldrich Chemie GmbH, Steinheim, Germany) was used for in vitro and in vivo studies. Amoxicillin trihydrate of known potency (GlaxoSmithKline S.A., Madrid, Spain) and amoxicillin sodium salt (Clamoxyl ® 1 g; GlaxoSmithKline S.A.) were used for in vitro and in vivo studies, respectively.

Minimum inhibitory concentrations and time-kill analysis

MICs of novobiocin and amoxicillin for the various strains were determined by the microdilution method approved by the Clinical and Laboratory Standards Institute (CLSI) [START_REF]Performance standards for antimicrobial susceptibility testing[END_REF] novobiocin or amoxicillin at 2, 8 or 32 MIC, except for the strain that was most resistant to penicillin (strain SPC2552) for which the amoxicillin concentration of 32 MIC was not tested owing to poor solubility of the powder.

Bacteria were grown in broth to early log phase and then inoculated tubes with CA-MHB-LHB were incubated at 35 C in normal atmosphere at a final titre of 2  10 6 colony-forming units (CFU)/mL. Samples of 100 L were removed at the time of inoculation (time zero) and after 2 h and 5 h, diluted and plated on blood agar plates for colony counting. The killing effects of antibiotics were thus compared by determining the change in log 10 CFU/mL at 2 h and 5 h compared with counts in the control tubes at each time point, and such differences in bacterial titres were expressed as negative values. Antibiotic carryover was minimised by dilution. Each test was performed in duplicate.

Mouse sepsis model

Experimental animals were adult female Swiss mice weighing 30  2 g (Centro de Investigaciones Biológicas, Madrid, Spain). Animals were housed in regulation cages with free access to food and water.

For animal inoculation, pneumococcal suspensions were prepared immediately before use by suspending colonies from fresh overnight cultures on 5% blood agar plates in sterile Todd-Hewitt broth (THB), adjusted to an optical density giving a concentration of ca. 5  10 8 CFU/mL. For the penicillin-susceptible A c c e p t e d M a n u s c r i p t 7 strain, the suspension was further diluted into THB to a final inoculum ranging from 4.8  10 2 to 1.3  10 4 CFU/mL. For the three penicillin-resistant strains and immediately before inoculation, the bacterial suspensions were diluted 1:10 in molten nutrient agar, which was used as adjuvant for inoculation of the mice, yielding a final agar concentration of 1.5% (w/v) and a final inoculum ranging from 4.2  10 7 to 1.4  10 8 CFU/mL. Sepsis with each strain was induced by intraperitoneal (i.p.) injection of 0.5 mL of inoculum into non-anaesthetised mice 1 h before therapy with antibiotics or apyrogen sterile distilled water (control).

These inocula were 80-100% lethal within 1-3 days and provided a consistently high number of bacteria in the peritoneal cavity. The inoculum size was confirmed by determination of the bacterial titre in each experiment. The potential lethal effect of agar alone was examined by i.p. injection of 0.5 mL of 1.5% (w/v) agar-THB solution to three animals. None of these animals appeared ill or died within the 72 h period of observation.

Treatment protocols

Treatment of mice was performed as a subcutaneous (s.c.) injection in a volume of 0.1 mL per dose in the neck region. For the efficacy studies, four doses of antibiotics were given at 1, 5, 24 and 48 h after infection, and survival was recorded daily throughout the study period, which ended 72 h after inoculation. At the end of the study, animals were euthanised by CO 2 overdose, and peritoneal lavage fluid specimens were collected and processed for CFU determination. Peritoneal lavage was also performed post-mortem in animals that were found dead before finishing the experiment. The results of peritoneal bacterial load were expressed as mean ± standard deviation (S.D.) log 10 A c c e p t e d M a n u s c r i p t 8 CFU/mL. Differences in the number of CFU/mL between treated and control animals were calculated to compare the effectiveness of the treatment regimens. The lower limit of organism quantification in these studies was 10 CFU/mL. Two types of experiments were performed to evaluate the effectiveness of antibiotic therapy, and the most effective antimicrobial regimen was defined as the regimen that achieved the highest survival rate and the most potent antibacterial effect in the peritoneal fluid.

Drug effectiveness in sepsis caused by a penicillin-susceptible strain

Mice with sepsis induced by the penicillin-susceptible strain (AR33118) received novobiocin (25, 50, 100 or 200 mg/kg) or amoxicillin (25 mg/kg). A group of mice received apyrogen sterile distilled water as control for the lethality and the peritoneal bacterial load of the infection. Mostly, five to seven mice were used for each regimen in these dose-ranging experiments.

Drug effectiveness in sepsis caused by penicillin-resistant strains

Following infection with each of the three penicillin-resistant S. pneumoniae strains, mice were treated with doses of novobiocin at 100 mg/kg or 200 mg/kg or amoxicillin at 50 mg/kg or 200 mg/kg. A group of mice that received apyrogen sterile distilled water was tested as control. Each regimen was given to mice in groups of at least seven animals (range, seven to ten animals). Survival was noted daily and peritoneal bacterial loads were determined as above.

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

Pharmacokinetics and tolerance of novobiocin

A single-dose plasma pharmacokinetic (PK) study of novobiocin was performed in non-infected mice given s.c. doses of novobiocin at 200 mg/kg. For each time point, three mice were sampled by cardiac puncture (immediately after euthanasia with CO 2 overdose) at 15, 30, 60, 120, 240, 360, 540 and 720 min after injection with heparin-rinsed syringes. Blood samples were centrifuged at 5000  g for 10 min. The plasma obtained was divided into two parts and one of them was centrifuged in tubes with filters with a cut-off of ca. 30 kDa (Centrifree ® YM-30; Millipore, Carrigtwohill, Ireland) in a fixed-angle rotor at 3000  g for 12 min. Both samples were stored at 20 C until analysis. Novobiocin concentrations were determined by an agar diffusion microbiological assay using Kocuria rhizophila (previously designated as Micrococcus luteus) ATCC 9341 as the test organism [START_REF] Tang | Reclassification of ATCC 9341 from Micrococcus luteus to Kocuria rhizophila[END_REF]. The antibiotic concentration in both parts (total plasma and protein-free ultrafiltrate) was calculated using standard curves derived from solutions prepared with different antibiotic concentrations dissolved, respectively, in pooled mouse plasma and 0.9% NaCl. Analyses were performed in duplicate and the intraday and interday variation was <10%.

PK analysis of novobiocin in total plasma and plasma ultrafiltrate was performed using TopFit V2 software (Karl Thomae, Boehringer-Ingelheim, Ingelheim, Germany) and a non-compartmental model. The area under the concentrationtime curve (AUC) of novobiocin from 0 min (time of administration) to 720 min was calculated using the linear trapezoidal rule.

A c c e p t e d M a n u s c r i p t 10 Four non-infected animals were injected subcutaneously with four doses of 200 mg/kg novobiocin at 1, 5, 24 and 48 h to monitor tolerance. Weight, aspect and behaviour of mice were examined daily for 1 week and weekly thereafter for 4 weeks. In addition, the aspect (once a week) and survival of mice were observed for 4 months.

Data analysis

Survival rates were compared by Fisher's exact test. Bacterial counts in peritoneal lavage of the different groups were presented as mean  S.D. and 95% confidence interval. Comparison of peritoneal bacterial counts between two groups was performed by Mann-Whitney test, and comparisons among three or more groups were examined with Kruskal-Wallis test followed by Dunn's multiple-comparisons test among groups when they were significantly different. Culture-negative samples (<10 CFU/mL) were included in the calculation of means assuming a value at the detection limit (1.0 log 10 CFU/mL).

A P-value of <0.05 was considered significant.

Results

Minimum inhibitory concentrations and time-kill curves

Novobiocin and amoxicillin MICs, respectively, of the study organisms were as follows: strain AR33118, 0.25 mg/L and 0.015 mg/L; strain MJD3693, 0.5 mg/L and 8 mg/L; strain SPC2162, 0.25 mg/L and 16 mg/L; and strain SPC2552, 0.5 mg/L and 64 mg/L. 1.

Baseline inocula ranged from 6.03 log 10 CFU/mL to 6.75 log 10 CFU/mL (time zero). Novobiocin exposure resulted in a >2 log 10 difference in viable counts versus the controls for all strains and all concentrations tested by 5 h, except for strains AR33118 and SPC2162 at 2 MIC. The antibacterial effect of amoxicillin was more potent and rapid than that of novobiocin, achieving at 2 MIC a >2 log 10 difference by 2 h and >4 log 10 difference by 5 h, but this effect was somewhat lower for the most resistant strain (SPC2552). As shown in Table 1, for novobiocin there was an increase in killing by increasing concentrations on MIC basis as well as on time basis by comparing 2 h vs. 5 h.

Therapeutic efficacy in sepsis caused by amoxicillin-susceptible andresistant strains

The in vivo efficacy of novobiocin against amoxicillin-susceptible and -resistant S. pneumoniae strains in a mouse sepsis model is shown in Fig. 1. With the treatment schedule consisting of four s.c. doses at 1, 5, 24 and 48 h after infection, both novobiocin 200 mg/kg and amoxicillin 25 mg/kg were associated with a 100% survival rate of mice infected with the amoxicillin-susceptible strain AR33118 (P < 0.01 vs. untreated control group). For this strain, novobiocin 100 mg/kg achieved a 71% survival rate.

With the amoxicillin-resistant strains, novobiocin 200 mg/kg achieved the highest protection (90-100% survivors), followed by amoxicillin 200 mg/kg (60-100%), and these differences were statistically significant (P < 0.01) compared A c c e p t e d M a n u s c r i p t 12 with the controls, except for the group infected with strain SPC2162 and treated with amoxicillin 200 mg/kg, which had a 60% survival rate. Novobiocin 100 mg/kg protected 50-87.5% of the animals, whereas amoxicillin 50 mg/kg protected only 14.3-25% of the animals, with the lowest survival rate in animals infected with the most resistant strain (SPC2552). Notably, against this strain the 200 mg/kg amoxicillin regimen gave protection (87.5% survivors) similar to that showed by novobiocin 100 mg/kg. Table 2 shows the peritoneal bacterial counts after therapy with novobiocin and amoxicillin regimens in mice with sepsis induced by four pneumococcal strains that were survivors and were humanely sacrificed for sampling at 72 h after infection or animals that died before 72 h. All control animals that died had >8 log 10 CFU/mL in the peritoneum (mean value 8.7 log 10 CFU/mL, range 8.2-9.1 log 10 CFU/mL).

All mice infected with the amoxicillin-susceptible strain (AR33118) and treated with both novobiocin 200 mg/kg and amoxicillin 25 mg/kg survived, and the bacterial load in the peritoneum was undetectable, whereas the mean decline in peritoneal bacterial counts of survivor mice treated with novobiocin 100 mg/kg compared with those of the control group was only 1.7 log units.

Bacterial clearance (undetectable CFU/mL) in peritoneal lavage fluid after sacrifice of survivor mice infected with the amoxicillin-resistant strains was detected as follows: (a) among mice infected with strain MJD3693, in five of nine animals treated with the high-dose novobiocin regimen, in two of five mice

Novobiocin pharmacokinetics and tolerance

Single-dose PK studies performed in non-infected mice that received single s.c. doses of 200 mg/kg novobiocin demonstrated the following PK values for total plasma and ultrafiltrate, respectively: elimination half-life, 151 min and 215 min; AUC 945.0 mg h/L and 136.6 mg h/L; and maximal concentration 147 mg/L and 18 mg/L. The level of protein binding in mouse plasma was 85%.

Long-term evaluation revealed appropriate tolerance to novobiocin at the tested regimen, as assessed by observing the weight, aspect and behaviour of the treated mice for 4 weeks, as well as the aspect and survival for 4 months.

Discussion

Multidrug-resistant and highly antibiotic-resistant pneumococci have emerged as significant pathogens causing community-acquired infections worldwide [START_REF] Richter | Changing epidemiology of antimicrobial-resistant Streptococcus pneumoniae in the United States, 2004-2005[END_REF]. However, following the 2008 CLSI parenteral penicillin breakpoints for nonmeningitis pneumococcal isolates (intermediate, 4 mg/L; resistant, 8 mg/L) [START_REF]Performance standards for antimicrobial susceptibility testing[END_REF], there are few up-to-date published studies dealing with highly -lactamresistant pneumococci (penicillin MIC ≥ 8 mg/L), especially regarding their optimal antibiotic therapy [START_REF] Mcgee | In vitro evaluation of the antimicrobial activity of ceftaroline against cephalosporin-resistant isolates of Streptococcus pneumoniae[END_REF][START_REF] Figueiredo | Novel penicillin-resistant clones of Streptococcus pneumoniae in the Czech Republic and in Slovakia[END_REF][START_REF] Soriano | Breakthrough in penicillin resistance? Streptococcus pneumoniae isolates with penicillin/cefotaxime MICs of 16 mg/L and their genotypic and geographical relatedness[END_REF]. Among therapeutic strategies for combating pneumococcal infections caused by highly -lactam-resistant strains, here we have explored the use of novobiocin, an old drug, which we hypothesise could be re-introduced to the antibiotic armamentarium specifically for therapy of pneumococcal infections caused by strains with high-level resistance to the clinically used antibiotics.

Novobiocin was extensively used before the development of broad-spectrum agents such as -lactam and quinolone agents. Because novobiocin displays potent activity against staphylococci, including meticillin-resistant Staphylococcus aureus (MRSA), pneumococci, enterococci and many other Gram-positive organisms [START_REF] Gombert | Susceptibility of multiply antibiotic-resistant pneumococci to the new quinoline antibiotics, nalidixic acid, coumermycin, and novobiocin[END_REF][START_REF] Jones | Should novobiocin be clinically re-evaluated?[END_REF], during the 1980s the suitability of clinical reevaluation of this drug was suggested [START_REF] Jones | Should novobiocin be clinically re-evaluated?[END_REF]. Clinical trials conducted in the 1990s demonstrated the efficacy of novobiocin-containing combinations for eradicating the carrier state of MRSA [START_REF] Arathoon | Efficacy of short courses of oral novobiocin-rifampin in eradicating carrier state of methicillinresistant Staphylococcus aureus and in vitro killing studies of clinical isolates[END_REF][START_REF] Walsh | Randomized double-blinded trial of rifampin with either novobiocin or trimethoprim-sulfamethoxazole against methicillin-resistant Staphylococcus aureus colonization: prevention of antimicrobial resistance and effect of host factors on outcome[END_REF]. Indeed, novobiocin combined with rifampicin prevented the development of resistance in MRSA to either drug even more efficiently than the combination of co-trimoxazole and rifampicin [START_REF] Walsh | Randomized double-blinded trial of rifampin with either novobiocin or trimethoprim-sulfamethoxazole against methicillin-resistant Staphylococcus aureus colonization: prevention of antimicrobial resistance and effect of host factors on outcome[END_REF].

Remarkably, among severely ill patients with haematological disorders, the A c c e p t e d M a n u s c r i p t combination of novobiocin with doxycycline or rifampicin was effective for the treatment of vancomycin-resistant Enterococcus faecium bacteraemia [START_REF] Montecalvo | Effect of novobiocin-containing antimicrobial regimens on infection and colonization with vancomycin-resistant Enterococcus faecium[END_REF].

Regarding the side effects associated with the use of novobiocin alone or in combination, it should be stressed that many patients [START_REF] Arathoon | Efficacy of short courses of oral novobiocin-rifampin in eradicating carrier state of methicillinresistant Staphylococcus aureus and in vitro killing studies of clinical isolates[END_REF][START_REF] Walsh | Randomized double-blinded trial of rifampin with either novobiocin or trimethoprim-sulfamethoxazole against methicillin-resistant Staphylococcus aureus colonization: prevention of antimicrobial resistance and effect of host factors on outcome[END_REF][START_REF] Shane | Novobiocin, a new antibiotic[END_REF] and healthy volunteers [START_REF] Drusano | Steady-state serum pharmacokinetics of novobiocin and rifampin alone and in combination[END_REF][START_REF] Welch | A study of the sensitizing potential of novobiocin[END_REF] received this drug, mostly at 500 mg every 12 h for a variable period of time, without reporting relevant toxicity, and it has been suggested that some of the side effects could be attributed to impurities in the formulation that have since been removed [START_REF] Walsh | Randomized double-blinded trial of rifampin with either novobiocin or trimethoprim-sulfamethoxazole against methicillin-resistant Staphylococcus aureus colonization: prevention of antimicrobial resistance and effect of host factors on outcome[END_REF]. Besides, novobiocin has also been administered as a modulator of alkylating agent cytotoxicity combined with cyclophosphamide, with the maximum tolerated dose being 6 g/day of novobiocin [START_REF] Eder | A phase I clinical trial of novobiocin, a modulator of alkylating agent cytotoxicity[END_REF].

The in vitro results using time-kill curves show that the antibacterial effect of amoxicillin is more potent and rapid than that of novobiocin but the concentrations of amoxicillin used (16-512 mg/L) for the amoxicillin-resistant strains were much higher than those of novobiocin (0.5-16 mg/L).

In the mouse sepsis model induced by an amoxicillin-susceptible pneumococcal strain, the highest dosage of novobiocin (200 mg/kg) was as effective as amoxicillin 25 mg/kg in terms of survival and killing of the peritoneal bacterial load. Results with the amoxicillin-resistant strains show that dosages of 200 mg/kg novobiocin or amoxicillin were needed to cure most animals.

Nevertheless, survival in animals inoculated with strain SPC2162 was, respectively, 100% and 60% when treated with 200 mg/kg of both novobiocin and amoxicillin. The doses selected for amoxicillin administration were based on the results from a previous study by our group [START_REF] Soriano | Correlation of in-vitro activity and pharmacokinetic parameters with in-vivo effect of amoxycillin, co-amoxiclav and cefotaxime in a murine model of pneumococcal pneumonia[END_REF] which showed that the maximum amoxicillin concentrations attained in mouse serum after single doses of amoxicillin of 200 mg/kg and 50 mg/kg were 300 mg/L and 75 mg/L, respectively. However, such high amoxicillin concentrations cannot be achieved in patients following oral administration of usual dosages. In fact, peak levels of amoxicillin of 7.5 mg/L and 4 mg/L in the blood of patients occur 2 h after ingestion of 500 mg and 250 mg doses taken fasting, respectively [START_REF] Mandell | Penicillins, cephalosporins, and other -lactam antibiotics[END_REF][START_REF] Neu | Principles and practice of infectious diseases[END_REF], so only a high dose of this drug administered parenterally could achieve a peak serum concentration closed to 100 mg/L. On the other hand, a PK study carried out in humans, as determined by HPLC, showed that after ingestion of novobiocin at 500 mg every 12 h the mean peak serum concentration was 62.5  13.4 mg/L with an AUC of 407 mg h/L and a half-life of ca. 6 h [START_REF] Drusano | Steady-state serum pharmacokinetics of novobiocin and rifampin alone and in combination[END_REF], but even higher concentrations can be achieved following parenteral administration. For a highly protein-bound drug such as novobiocin, free drug concentrations must be determined, and our results show that maximum survival of animals was achieved with a free peak plasma concentration of ca. 18 mg/L. Our results also show that novobiocin could achieve sufficient concentrations to eradicate highly -lactam-resistant pneumococcal strains, whereas amoxicillin does not.

Lastly, to our knowledge there is insufficient experimental evidence on the development of novobiocin resistance in pneumococci to preclude its use for the therapy of multidrug-resistant pneumococcal infections. Very few studies have approached this problem in S. pneumoniae. An in vitro study by Muñoz et In conclusion, until novel antibacterial agents are discovered, our in vitro and in vivo data suggest that novobiocin may be a therapeutic alternative in pneumococcal infections caused by highly -lactam-resistant strains. a Novobiocin and amoxicillin MICs (mg/L), respectively.

b Differences in bacterial colony counts between those found in antibiotic-containing tubes and those shown in control tubes at 2 h and 5 h (inocula ranged from 6.03 log 10 CFU/mL to 6.75 log 10 CFU/mL at time zero).

  needed against infections caused by highly multidrug-resistant Streptococcus pneumoniae. Novobiocin, an old antibiotic, was tested in vitro and in a murine sepsis model against one amoxicillinsusceptible and three amoxicillin-resistant strains [minimum inhibitory concentrations (MICs) 8-64 mg/L].

  using cation-adjusted Mueller-Hinton II broth (Becton, Dickinson and Co., Le Pont-de-Claix, France) supplemented with 4% lysed horse blood (CA-MHB-LHB). Modal values of three separate determinations were considered.

  assays were performed by exposing cultures of the various strains to

  data for the four S. pneumoniae strains are presented in Table

  low-dose novobiocin regimen, and in seven of eight mice treated with the high-dose amoxicillin regimen; (b) among mice infected with strain SPC2162, in six of nine mice treated with novobiocin 200 mg/kg, in two of four mice treated with novobiocin 100 mg/kg, and in three of six mice treated with amoxicillin 200 mg/kg; and (c) among mice infected with strain SPC2552, in seven of eight mice receiving novobiocin 200 mg/kg, in two of seven mice receiving novobiocin 100 mg/kg, and in all seven mice receiving amoxicillin 200 mg/kg. Overall, the killing effect of the different antibiotic regimens in the peritoneum in mice infected with the amoxicillin-resistant strains could be ranked as follows (mean log 10 CFU/mL between treated and untreated control mice after 72 h): novobiocin 200 mg/kg (6.6) > amoxicillin 200 mg/kg (5.6) > novobiocin 100 mg/kg (3.7) > amoxicillin 50 mg/kg (0.7).

A

  c c e p t e d M a n u s c r i p t al.[START_REF] Muñoz | Ser-127-to-Leu substitution in the DNA gyrase B subunit of Streptococcus pneumoniae is implicated in novobiocin resistance[END_REF] described that a specific amino acid change (Ser-127Leu) in the DNA gyrase B subunit of S. pneumoniae increases novobiocin resistance, with MICs of 1 mg/L to 128 mg/L for the novobiocin-susceptible and -resistant strain, respectively.

  Animal studies were approved by the Animal Experimentation Ethics Committee of the Fundación Jiménez Díaz (Madrid, Spain). All animal studies were conducted in accordance with regulations regarding the care and use of laboratory animals in the European Union.

Fig. 1 .

 1 Fig. 1. Survival of mice with sepsis infected with four different Streptococcus

A c c e p t e d M a n u s c r i p t 1 2 4 

 124 Table Bacterial counts (mean  standard deviation) in peritoneal fluid and survival after therapy with novobiocin and amoxicillin regimens in mice with sepsis induced by four Streptococcus pneumoniae strains a Drug regimen b Log 10 CFU/mL in survivor/log 10 CFU/mL in dead animals (no. of survivor/no. of dead mice) 9.1  0.4 (0/6) 3.3/9.1  0.7 (1/9) 3.4  1.7/8.2  0.8 (2/8) None d /8.3  0.6 (01.5/8.5  0.7 (5/2) 5.6  4.3/7.2  0.2 (5/3) 3.5  2.4/6.7  2.8 (4/4) 2.9  1.5 */5.7 (7/1) 200 mg/kg <1.0 */None d (5/0) 2.0  1.3 */6.2 (9/1) 1.8  1.7 */None d (9/0) 1.5  1.4 */None d (8 3.5/9.2  0.8 (2/6) 5.2  4.8/8.0  0.5 (2/6) <1.0/8.9  0.0 (1/6) 200 mg/kg NT 1.4  1.2 */None d (8/0) 3.7  3.1/8.2  0.8 (6/4) <1.0 */7.3 (7/1) NT, not tested. a The respective novobiocin/amoxicillin MICs of strains AR33118, MJD3693, SPC2162 and SPC2552 were 0.25/0.015, 0.5/8, 0.25/16 and 0.5/64 mg/L.

Table 1

 1 Time-kill experiments of four Streptococcus pneumoniae strains after exposure to novobiocin or amoxicillin at 2, 8 and 32 times the 0.68 ± 0.21 1.36 ± 0.16 1.45 ± 0.04 3.96 ± 0.12 4.18 ± 0.20 3.88 ± 0.11 5 1.29 ± 0.05 2.71 ± 0.51 3.04 ± 0.15 4.33 ± 1.42 4.36 ± 0.32 5.80 ± 1.01 MJD3693 (0.5, 8) 2 1.31 ± 0.06 1.58 ± 0.16 1.72 ± 0.08 2.58 ± 0.16 3.16 ± 0.03 3.18 ± 0.01 5 2.89 ± 0.05 3.35 ± 0.1 3.50 ± 0.01 4.73 ± 0.09 4.68 ± 0.14 4.68 ± 0.35 SPC2162 (0.25, 16) 2 1.20 ± 0.16 1.53 ± 0.14 1.75 ± 0.10 2.46 ± 0.00 2.68 ± 0.09 2.17 ± 0.15 5 1.96 ± 0.01 3.13 ± 0.10 3.74 ± 0.25 4.64 ± 0.33 4.66 ± 0.04 3.62 ± 0.03 SPC2552 (0.5, 64) 2 1.55 ± 0.14 1.90 ± 0.5 2.36 ± 0.23 2.55 ± 0.28 1.85 ± 0.02 NT 5 2.98 ± 0.33 3.59 ± 0.03 4.17 ± 0.12 4.10 ± 0.67 3.64 ± 0.21 NT CFU, colony-forming units; NT, not tested.

	minimum inhibitory concentration (MIC)
	Strain (MIC a )	Time (h) Differences in log 10 CFU/mL b
		Novobiocin			Amoxicillin
		2 MIC	8 MIC	32 MIC	2 MIC	8 MIC	32 MIC
	AR33118 (0.25, 0.015) 2

b Doses were given at 1, 5, 24 and 48 h after infection.c Evaluation was performed at 72 h after the start of therapy or before if the animal was found dead.d No bacterial counts are shown because there were no animals in these groups.

Funding

Research contracts were received by VR-C (COMBACT-CM, S-BIO-0260/2006) and by GdP (CPI/0305/2 007 attached to COMBACT-CM), both from Comunidad Autónoma de Madrid, Spain. VR-C is currently supported by a research contract from the Subprogram Juan de La Cierva (JCI-2008-02690; Ministerio de Ciencia e Innovación Tecnológica, Spain). LH received a grant from the Fundación Conchita Rábago (Madrid, Spain), and PN from the Alan program (European Union). This work was performed under the Research Collaboration Agreement between the Consejo Superior de Investigaciones Científicas and the Fundación Jiménez Díaz.

Competing interests

None declared.

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