Ranjith Rajendran

Ryan F Quinn

Colin Murray

Elaine Mcculloch

Craig Williams

Gordon Ramage
email: g.ramage@dental.gla.ac.uk

Efflux pumps may play a role in tigecycline resistance in Burkholderia species

Keywords:

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

ABSTRACT

The purpose of this study was to investigate the role of multidrug resistance efflux pumps in relation to decreased susceptibility to tigecycline in clinical isolates of Burkholderia cepacia complex (BCC). The role of efflux pumps was analysed using the efflux pump inhibitor (EPI) MC-207,110. Minimum inhibitory concentrations (MICs) were determined for each strain against tigecycline alone and in the presence of 64 mg/L MC-207,110. The effect of efflux pump inhibition on the susceptibility of BCC isolates to tigecycline was assessed by a checkerboard titration assay. Ala-Nap uptake assay was performed to determine efflux pump activity in different strains. The checkerboard titration assay showed that the MIC decreased with increasing concentrations of EPI. MICs for tigecycline in the clinical isolates ranged between 8 mg/L and 32 mg/L, whereas in the presence of MC-207,110, MICs decreased significantly (range <0.125-1.0 mg/L; 16 to >256 times reduction). Efflux pump activity was shown to be greatest in strains with the highest MIC and vice versa. In conclusion, BCC possess efflux pumps that influence their resistance to tigecycline. Use of an inhibitor of these pumps restored sensitivity to the antibiotic. Therefore, a combination of tigecycline and EPI to augment its efficacy may present an attractive therapeutic option.

Introduction

Gram-negative microorganisms such as Pseudomonas aeruginosa and Burkholderia cepacia are clinically important pathogens of the cystic fibrosis (CF) lung. A hallmark of these infections is failure to eradicate the organism because of high-level multiple drug resistance [START_REF] Poole | Multidrug resistance in Gram-negative bacteria[END_REF], which is known to emerge during treatment [START_REF] Davies | Emerging and unusual Gram-negative infections in cystic fibrosis[END_REF]. Tigecycline, a novel expanded broad-spectrum glycylcycline antibiotic, has been shown to be active against a broad range of Gram-negative, Gram-positive, anaerobic and atypical bacteria [START_REF] Bouchillon | In vitro evaluation of tigecycline and comparative agents in 3049 clinical isolates: 2001 to 2002[END_REF]. However, its activity against P. aeruginosa is poor, with the microorganism's inherent resistance attributed to the activity of broad-substrate efflux pumps [START_REF] Dean | Efflux-mediated resistance to tigecycline (GAR-936) in Pseudomonas aeruginosa PAO1[END_REF]. The activity of tigecycline against B. cepacia complex (BCC) has also been reported to be poor [START_REF] Milatovic | Activities of the glycylcycline tigecycline (GAR-936) against 1,924 recent European clinical bacterial isolates[END_REF], but the mechanisms of resistance are poorly defined.

Drug efflux pump expression is a common resistance mechanism leading to high levels of drug resistance as a result of apparent synergism with the atypically impermeable outer membrane, thus limiting influx of antimicrobial agents.

However, the action of these pumps may be blocked using efflux pump inhibitors (EPIs), compounds that bind within the specific substrate pocket and inhibit efflux pump activity [START_REF] Marquez | Bacterial efflux systems and efflux pumps inhibitors[END_REF]. MC-207,110 is the first identified broad-spectrum EPI that effectively inhibits all clinically relevant efflux pumps in Gram-negative bacteria [START_REF] Pages | Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria[END_REF][START_REF] Pages | Inhibitors of efflux pumps in Gram-negative bacteria[END_REF]. The aim of this study was to determine whether tigecycline resistance in BCC is medicated by efflux pumps and, if so, whether EPIs can be used to augment the efficacy of the glycylcycline molecule.

Materials and methods

Bacterial strains and media

Burkholderia strains used in this study (Table 1) were isolated from CF patients at the Royal Hospital for Sick Children (Yorkhill Division), Glasgow, UK. All strains were maintained and grown on Luria broth or agar at 37 C. Mueller-Hinton broth was used for all susceptibility testing assays.

Susceptibility testing

Tigecycline white powder was kindly provided by Wyeth Pharmaceuticals (Princeton, NJ) and the EPI L-Phe-L-Arg--naphthylamide (MC-207,110) was purchased from Sigma-Aldrich (Gillingham, UK). These were prepared freshly at working concentrations in ddH 2 O when required. The minimum inhibitory concentration (MIC) of tigecycline was determined by standard Clinical and Laboratory Standard Institute (CLSI) broth microdilution methodology in 96-well microtitre plates. A checkerboard assay was employed to show the effect of different concentrations of MC-207,110 on the MIC of tigecycline, as described previously by Lomovskaya et al. [START_REF] Lomovskaya | Identification and characterization of inhibitors of multidrug resistance efflux A c c e p t e d M a n u s c r i p t 11 pumps in Pseudomonas aeruginosa: novel agents for combination therapy[END_REF]. This was undertaken using five strains encompassing the different genomovars (given in parenthesis), including K56-2 A c c e p t e d M a n u s c r i p t 5 (III), YHBCC1 (II), YHBCC13 (IV), YHBCC14 (V) and YHBG1. Tigecycline was tested at a range from 0.06 mg/L to 64 mg/L in combination with MC-207,110 at a range of 8 mg/L to 512 mg/L. Viability was assessed using an Alamar Blue ® colorimetric assay as per the manufacturer's instructions (Invitrogen, Paisley, UK). Following these initial experiments, a defined concentration of MC-207,110 was selected (64 mg/L) for subsequent microdilution testing on the entire panel of strains. This was based upon data obtained from the checkerboard assay, which showed no antimicrobial activity at the concentrations tested.

Ala-Nap uptake assay

Efflux pump activity was assessed using an Ala-Nap fluorescent assay as previously described [START_REF] Lomovskaya | Identification and characterization of inhibitors of multidrug resistance efflux A c c e p t e d M a n u s c r i p t 11 pumps in Pseudomonas aeruginosa: novel agents for combination therapy[END_REF]. Ala-Nap is enzymatically cleaved inside cells to produce highly fluorescent -naphthylamine, thus the higher the fluorescence the lower the efflux pump activity. Selected isolates were washed and standardised to 5 

Results

The sensitivity of 17 different strains of BCC and 3 Burkholderia gladioli to tigecycline was evaluated by broth microdilution according to CLSI methodology.

The MIC 50 and MIC 90 (MIC for 50% and 90% of the isolates, respectively) for BCC were 32 mg/L (range 8-32 mg/L) (Table 1). Burkholderia gladioli strains were more sensitive (range 4-8 mg/L). For EPI studies, the MIC value of MC-207,110 was determined to be >512 mg/L for all the strains tested. To assess the effect of inhibition of efflux pumps upon the susceptibility of BCC, a standard checkerboard titration assay was performed on five strains. An inverse relationship between tigecycline and MC-207,110 concentration was observed, i.e. the MIC of tigecycline decreased with an increase in the concentration of MC-207,110 (Fig. 1), indicating that MC-207,110 at a concentration of 64 mg/L exhibited a 50% potentiating effect on a decrease in the MIC. This concentration exhibited no detrimental effect on bacterial viability when assessed using an Alamar Blue ® assay. This concentration was therefore used for all subsequent experiments using standard CLSI broth microdilution methods. The MICs of tigecycline in the presence of MC-207,110 were determined for each BCC strain and it was found that the MIC 50 and MIC 90 values were reduced to 0.5 mg/L and 1 mg/L, respectively, with a range of <0.125 mg/L to 1 mg/L (Table 1). The level of MIC reduction ranged from 16 to >256 times for both BCC and B. gladioli. Subsequent analysis of -naphthylamine production by three strains demonstrated differential efflux activity (Fig. 2). The rate of cleavage of Ala-Nap was highest in YHBG3 (MIC = 8 mg/L; MIC reduction >64) compared with

Discussion

The increasing prevalence of resistant bacteria demands that a renewed effort be made to seek new and alternative antibiotics effective against drug-resistant pathogens. Tigecycline is a novel antibiotic developed to overcome widespread bacterial resistance to tetracycline and minocycline. It is effective against many Gram-positive and Gram-negative microorganisms but has little activity against the non-fermenting CF pathogens P. aeruginosa and Burkholderia spp. The mechanism of resistance has been clearly demonstrated for P. aeruginosa, but in BCC it remains to be determined [START_REF] Dean | Efflux-mediated resistance to tigecycline (GAR-936) in Pseudomonas aeruginosa PAO1[END_REF][START_REF] Guglierame | Efflux pump genes of the resistance-nodulation-division family in Burkholderia cenocepacia genome[END_REF]. The data presented here indicate a role for efflux pumps.

The effect of efflux pumps was studied by inhibiting those systems using a broadspectrum EPI MC-207,110, a substrate of efflux pumps. Previous studies have demonstrated MC-207,110 to be a potential inhibitor of efflux pumps in Gramnegative bacteria such as P. aeruginosa as well as potentiating the activities of levofloxacin and chloramphenicol [START_REF] Pages | Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria[END_REF][START_REF] Lomovskaya | Identification and characterization of inhibitors of multidrug resistance efflux A c c e p t e d M a n u s c r i p t 11 pumps in Pseudomonas aeruginosa: novel agents for combination therapy[END_REF][START_REF] Bina | Effect of the efflux inhibitors 1-(1naphthylmethyl)-piperazine and phenyl-arginine--naphthylamide on antimicrobial susceptibility and virulence factor production in Vibrio cholerae[END_REF]. However, according to Chan et al. [START_REF] Chan | BpeAB-OprB, a multidrug efflux pump in Burkholderia pseudomallei[END_REF], MC-207,110 did not have any potentiating effect on the efflux of erythromycin or streptomycin by the BpeAB-OprM pump in Burkholderia pseudomallei. It can be concluded from these studies that MC-207,110 works as an active competitor of some, but not all, antibacterial drugs. Efflux pumps were A c c e p t e d M a n u s c r i p t 8 shown to be the mechanism of tigecycline resistance in Acinetobacter baumannii, which was shown to be reversible with MC-207,110 [START_REF] Peleg | Tigecycline efflux as a mechanism for nonsusceptibility in Acinetobacter baumannii[END_REF]. Moreover, a study by Hirakata et al. [START_REF] Hirakata | Efflux pump inhibitors reduce the invasiveness of Pseudomonas aeruginosa[END_REF] reported that in addition to its role as an inhibitor of efflux pump activity, it was also capable of reducing the invasiveness of P. aeruginosa PA01.

In this study, we report that MC-207,110 augmented the effect of tigecycline in a concentration-dependent manner in members of the BCC, demonstrating that these microorganisms may utilise efflux pumps as a resistance mechanism. As the concentration of EPI increased, the MIC to tigecycline was reduced, indicating the concentration-dependent competition for the extrusion of tigecycline through active efflux. The potency (MIC 90 = 32 mg/L) of tigecycline in the absence of MC-207,110 was similar to the results published by Milatovic et al. [START_REF] Milatovic | Activities of the glycylcycline tigecycline (GAR-936) against 1,924 recent European clinical bacterial isolates[END_REF], but in the presence of MC-207,110 the MICs decreased significantly (MIC 90 = 1 mg/L). Direct assessment of the efflux activity demonstrated a correlation between efflux activity and high MIC values, and conversely the lower MICs of B. gladioli were correlated with low efflux pump activity (Fig. 2).

Therefore, this supports the hypothesis that members of the BCC utilise efflux pumps for resistance to antibiotics.

In conclusion, CF pathogens of the BCC possess efflux pump proteins that have evolved to become integral to their natural physiological function and that also confer inherent resistance to tigecycline. However, these pumps can be

10 5

 5 cells in buffer solution [K 2 HPO 4 (50 mM), MgSO 4 (1 mM) and glucose (0.4%)] at pH 7.0 and dispensed into black, flat-bottomed microtitre plate (Costar ® 3603;Corning-Costar Corp., Corning, NY). The reaction was initiated by the addition of Ala-Nap at a final concentration of 128 mg/L. Fluorescence was quantified at 30s intervals for 1 h at 37 C using a fluorescent plate reader (FLUOstar OPTIMA; BMG LABTECH, Aylesbury, UK) with an excitation wavelength of 320 nm and an emission wavelength of 460 nm.

  = 16 mg/L; MIC reduction = 32) and YHBCC12 (MIC = 32 mg/L; MIC reduction >256), i.e. higher MICs correlated with greater efflux.

12 Fig. 1 .

 121 Fig. 1. Schematic representation of a checkerboard assay of Burkholderia

Fig. 2 .

 2 Fig. 2. Ala-Nap (128 mg/L) uptake of selected strains YHBG3, YHBCC4 and

Table 1

 1 Minimum inhibitory concentrations (MICs) of tigecycline in the presence and absence of the efflux pump inhibitor MC-207,110 (64 mg/L)

	Edited Table 1
	Strain	Tigecycline MIC (mg/L)	Fold
		Tigecycline	Tigecycline plus MC-	change
		alone	207,110
	Burkholderia cepacia complex (genomovar)
	K56-2 (III)	32	0.5	64
	ATCC 17765	32	1	32
	(III)
	YHBCC1 (II)	32	1	32
	YHBCC2 (II)	32	1	32
	YHBCC3 (II)	32	0.5	64
	YHBCC4 (II)	16	0.5	32
	YHBCC5 (III)	16	0.25	64
	YHBCC6 (III)	32	1	32
	YHBCC7 (III)	32	1	32
	YHBCC8 (III)	32	1	32
	YHBCC9 (III)	16	0.25	64
	YHBCC10 (III)	32	1	32
	YHBCC11 (III)	16	0.5	32
	YHBCC12 (III)	32	<0.125	>256
	YHBCC13 (IV)	8	0.5	16
	YHBCC14 (V)	16	0.25	64
	YHBCC15 (V)	16	1	16
	Burkholderia gladioli
	YHBG1	8	0.25	32
	YHBG2	4	0.25	16
	YHBG3	8	<0.125	>64

Funding

This work was supported by an unrestricted research grant from Wyeth Pharmaceuticals (Princeton, NJ).

A c c e p t e d M a n u s c r i p t 9 inactivated using competitive inhibitors to restore the activity of tigecycline.

Whether EPIs can be used for combination therapy with tigecycline in CF patients remains to be seen. Tigecycline is derived from a tetracycline that has been shown to have the capacity to modulate inflammatory processes. Using EPIs in combination with tigecycline in CF patients may provide the opportunity for an antibiotic with unrestricted broad-spectrum antimicrobial activity whilst also suppressing inflammation of the CF lung. Further studies are ongoing to investigate tigecycline further in this respect.

Competing interests

None declared.

Ethical approval

Not required.

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


Jacqueline Chevalier

Abdallah Mahamoud

Milad Baitiche

Elissavet Adam

Miguel Viveiros

Adriana Smarandache

Andra Militaru

Mihail L Pascu

Leonard Amaral

Jean-Marie Pagès
email: jean-marie.pages@univmed.fr

Quinazoline derivatives are efficient chemosensitizers of antibiotic activity in Enterobacter aerogenes, Klebsiella pneumoniae and Pseudomonas aeruginosa resistant strains

Keywords:

These authors contributed equally to this work. 2 Present address: Laboratoire des Matériaux Polymériques Multiphasiques (LMPMP), Faculté des Sciences de l'ingénieur, Université Ferhat Abbas, Sétif, Algérie.

A c c e p t e d M a n u s c r i p t 3 ABSTRACT Among the three series of quinazoline derivatives synthesised and studied in this work, some molecules increase the antibiotic susceptibility of Gram-negative bacteria presenting multidrug-resistant phenotypes. N-alkyl compounds induced an increase in the activity of chloramphenicol, nalidixic acid and sparfloxacin, which are substrates of the AcrAB-TolC and MexAB-OprM efflux pumps in clinical isolates.

These molecules are able to increase the intracellular concentration of chloramphenicol in efflux pump-overproducing strains. Their activity depends on the antibiotic structure, suggesting that different sites may be involved for the recognition of substrates by a given efflux pump. Quinazoline molecules exhibiting a nitro functional group are more active, and structure-activity relationship studies may be undertaken to identify the pharmacophoric group involved in the AcrB and MexB affinity sites.

Introduction

Chemoresistance is an important health challenge concerning infectious diseases, and the worldwide dissemination of multidrug-resistant (MDR) bacteria has severely compromised the efficacy of our antimicrobial weapons and has dramatically increased the frequency of therapeutic failure [START_REF] Falagas | Pandrug-resistant Gram-negative bacteria: the dawn of the post-antibiotic era?[END_REF]. The polyspecificity of bacterial efflux transporters that extrude antibiotics prior to reaching their intended target confers a general resistance phenotype that can enhance the acquisition of additional mechanisms of antibiotic resistance [START_REF] Li | Efflux-mediated drug resistance in bacteria: an update[END_REF][START_REF] Poole | Efflux pumps as antimicrobial resistance mechanisms[END_REF]. It has recently been demonstrated that expression of the AcrAB-TolC pump belonging to the resistancenodulation-cell division (RND) transporter family plays an important role in the selection of target mutations in resistant Gram-negative bacteria [START_REF] Li | Efflux-mediated drug resistance in bacteria: an update[END_REF][START_REF] Poole | Efflux pumps as antimicrobial resistance mechanisms[END_REF][START_REF] Pagès | Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria[END_REF]. Because of the clear involvement of RND transporters in the increased frequency of MDR clinical bacteria, efflux pumps are now considered as an attractive target for the development of antibiotic/efflux pump inhibitor (EPI) combinational therapy, similar to the current use of a -lactam/-lactamase inhibitor mix [START_REF] Pagès | Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria[END_REF]. Diverse compounds have been reported to alter the efflux activity of Gram-negative bacteria. Several strategies have been described for the selection of chemosensitizers that block the drug efflux capability of bacterial cells and reverse initial resistance to specific antibiotics [START_REF] Pagès | Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria[END_REF].

Using a screening test that measures the response of antibiotic-resistant

Enterobacter aerogenes strains, various compounds exhibiting some structural similarities to the quinolone family have been shown to restore the activity of antibiotics that are expelled by bacterial efflux pumps [START_REF] Pagès | Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria[END_REF]. These quinoline derivatives are mainly active on MDR clinical isolates that overexpress efflux pumps. It has been The purpose of the present study was: (i) to assess the ability of new compounds to restore drug susceptibility of E. aerogenes, Klebsiella pneumoniae and Pseudomonas aeruginosa MDR strains; and (ii) to determine the structure-activity relationships of these compounds. Three compounds were shown to exhibit a very efficient chemosensitizer profile and to restore significant susceptibility of various resistant clinical strains to structurally unrelated antibiotics.

Materials and methods

Chemistry

The synthetic pathways of the quinazoline derivatives have been reported previously [START_REF] Baïtiche | Synthesis of new quinazoline derivatives[END_REF].

Bacterial strains, growth conditions and antibiotic susceptibility testing

Enterobacter aerogenes CM-64, which is a chloramphenicol-resistant strain (ATCC 13048 derivative) overexpressing the AcrAB pump, and isolate EA27 exhibiting norfloxacin and chloramphenicol efflux were used [START_REF] Pagès | Efflux pump, the masked side of -lactam resistance in Klebsiella pneumoniae clinical isolates[END_REF][START_REF] Lorenzi | Geraniol restores antibiotic activities against multidrug-resistant isolates from Gramnegative species[END_REF]. Two K. pneumoniae isolates (KP4 and KP55) exhibiting active efflux of norfloxacin and chloramphenicol [START_REF] Pagès | Efflux pump, the masked side of -lactam resistance in Klebsiella pneumoniae clinical isolates[END_REF][START_REF] Lorenzi | Geraniol restores antibiotic activities against multidrug-resistant isolates from Gramnegative species[END_REF] as well as one strain of P. aeruginosa PA124 overexpressing OprM, the outer membrane channel of two efflux systems, were used [START_REF] Pagès | Efflux pump, the masked side of -lactam resistance in Klebsiella pneumoniae clinical isolates[END_REF][START_REF] Lorenzi | Geraniol restores antibiotic activities against multidrug-resistant isolates from Gramnegative species[END_REF]. Enterobacter aerogenes ATCC 13048, K. pneumoniae ATCC 11296 and P. aeruginosa PA01 were used as reference strains.

A c c e p t e d M a n u s c r i p t 6 To determine minimal inhibitory concentrations (MICs), ca. 10 6 cells growing in Mueller-Hinton (MH) broth at 37 C were inoculated into 1 mL of MH broth containing two-fold serial dilutions of each antibiotic. Results were read after 18 h at 37 C according to the guidelines of the Clinical and Laboratory Standards Institute [START_REF]Performance standards for antimicrobial susceptibility testing. Fourteenth informational supplement[END_REF]. To study synergistic activity, various concentrations of the potential inhibitors were added during incubation with the antibiotics [START_REF] Pagès | Efflux pump, the masked side of -lactam resistance in Klebsiella pneumoniae clinical isolates[END_REF][START_REF] Lorenzi | Geraniol restores antibiotic activities against multidrug-resistant isolates from Gramnegative species[END_REF]. Control experiments were carried out without the different inhibitors.

Chloramphenicol uptake

Exponential-phase bacteria grown in Luria-Bertani broth were pelleted, washed once and suspended to a density of 10 10 colony-forming units/mL in sodium phosphate buffer (50 mM, pH 7) containing 5 mM MgCl 2 .

Determination of compound stability

Stability studies were performed on compound 1188. The concentration of 1188 in solution ranged from 10 -3 M to 10 -6 M at pH 6.7. Solutions were kept in the dark and two cases were observed: (i) samples were kept at 4 C and then measured at room temperature (22 C) (when measured, they were first incubated for 30 min to reach room temperature); and (ii) samples were kept at room temperature. To rule out possible precipitation, the solutions were exposed to ultrasonic treatment (exposure time up to 15 min at 35 kHz with a peak output power of 240 W and HF power = 30 W eff ) [START_REF] Pascu | In vivo studies of the effects of alkyl substituted benzo[b]pyridinium compounds exposed to optical radiation[END_REF]. Stability was checked by ultraviolet-visible absorption spectra measurements [START_REF] Pascu | In vivo studies of the effects of alkyl substituted benzo[b]pyridinium compounds exposed to optical radiation[END_REF]. The stability of EPIs is a key point that has been reported by several studies focusing on molecules belonging to the peptidomimetic class [START_REF] Pagès | Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria[END_REF].

Absorption measurements performed between 2  10 -3 M and 10 -6 M allowed computation of a polymerisation degree of 1.018, thereby indicating that at this concentration range there were insignificant changes in the stability of the compound during the tested period.

Results

Effect of quinazoline compounds on various Enterobacter aerogenes strains

Enterobacter aerogenes ATCC 13048, the chloramphenicol-resistant variant CM-64 and a clinical isolate EA27 [START_REF] Pagès | Efflux pump, the masked side of -lactam resistance in Klebsiella pneumoniae clinical isolates[END_REF][START_REF] Lorenzi | Geraniol restores antibiotic activities against multidrug-resistant isolates from Gramnegative species[END_REF] were used to determine the intrinsic biological activity of various quinazolines (Table 1). The MICs of these compounds were especially high and were similar to that obtained with PAN, an EPI used for the determination of efflux-mediated antibiotic resistance in P. aeruginosa and E. aerogenes strains. Compounds were assayed at concentrations that have no intrinsic effect on their ability to decrease the level of chloramphenicol resistance of E. aerogenes EA27 and CM-64 expressing active efflux of chloramphenicol. Among these compounds, 1187, 1188, 1189 and 1190 were particularly effective as enhancers of chloramphenicol activity and reduced the MIC of chloramphenicol (Table 1). Although the individual responses of strains EA27 and CM-64 to each of the agents evaluated for an MIC, including the PAN control, was identical, the degree of susceptibility to chloramphenicol is far greater for the CM-64 strain than for the EA27 strain. The activity of compounds for reducing susceptibility to chloramphenicol differed between the two strains and did not appear to be related to the degree of resistance to chloramphenicol. This is to be expected since the two strains have been shown to differ remarkably with respect to their membrane physiology characteristics [START_REF] Pagès | Efflux pump, the masked side of -lactam resistance in Klebsiella pneumoniae clinical isolates[END_REF][START_REF] Lorenzi | Geraniol restores antibiotic activities against multidrug-resistant isolates from Gramnegative species[END_REF].

Determination of the MIC for each compound is an absolute requirement that assures

To further study the EPI-like effect, molecules having the greatest effect on reduction of the MIC of chloramphenicol were evaluated for their ability to increase the susceptibility of ATCC 13048 and CM-64 to various quinolones. Whereas a great reduction of the MICs of nalidixic acid and ciprofloxacin was produced by 1188 and 1189 against CM-64, these compounds produced a lesser but significant reduction of the MIC of norfloxacin (Table 2). These compounds had little, if any, effect on the MIC of nalidixic acid, norfloxacin, sparfloxacin or erythromycin against the ATCC 13048 reference strain.

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

Effect of quinazoline derivatives on chloramphenicol accumulation in Enterobacter aerogenes EA27 and CM-64

The effect of the quinazoline derivatives on intracellular accumulation of radiolabelled chloramphenicol by the chloramphenicol-resistant EA27 and CM-64 strains overexpressing the AcrAB-TolC efflux system [START_REF] Pagès | Efflux pump, the masked side of -lactam resistance in Klebsiella pneumoniae clinical isolates[END_REF][START_REF] Lorenzi | Geraniol restores antibiotic activities against multidrug-resistant isolates from Gramnegative species[END_REF] was evaluated. Controls with PAN, the well known EPI [START_REF] Pagès | Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria[END_REF], were conducted at the same time. Compounds 1187, 1188, 1189 and 1190 increased the level of intracellular chloramphenicol (Fig. 1).

The effect of 1188 on intracellular accumulation of chloramphenicol by EA27 and CM-64 strains was dose-dependent, as was the case for the positive control PAN (data not shown). It is interesting to check whether the quinazoline molecules are also able to increase the intracellular accumulation of another substrate of the AcrAB efflux pump. For this aim, the method recently developed to study ethidium bromide transport [START_REF] Viveiros | Demonstration of intrinsic efflux activity of Escherichia coli K-12 AG100 by an automated ethidium bromide method[END_REF] was performed with E. aerogenes EA27 resistant strain. The result indicates that addition of 1189 significantly increased the intracellular concentration of ethidium bromide in this strain that overproduces the AcrAB pump (Fig. 1).

Effect of quinazoline derivatives on other Gram-negative resistant bacteria

Various resistant strains of K. pneumoniae and P. aeruginosa [START_REF] Pagès | Efflux pump, the masked side of -lactam resistance in Klebsiella pneumoniae clinical isolates[END_REF][START_REF] Lorenzi | Geraniol restores antibiotic activities against multidrug-resistant isolates from Gramnegative species[END_REF] were tested with the compounds in combination with different classes of antibiotics. An efficient increase of susceptibility to chloramphenicol and nalidixic acid was obtained with the compounds against K. pneumoniae KP4 isolate (Table 2). For KP55 isolate, the activity of chloramphenicol was improved, whereas a lesser effect was evident for nalidixic acid activity owing to the presence of target mutations. The compounds produced a similar reduction in the MIC of tigecycline against the two isolates (KP55 and KP4). This suggests that the compounds are also effective in reducing antibiotic A c c e p t e d M a n u s c r i p t 10 resistance of K. pneumoniae resistant isolates. It is interesting to note that the level of restoration of antibiotic susceptibility depends on the antibiotic class (related to their structural and functional properties) and the presence of additional mechanisms of resistance such as target mutation that can modulate the effect [START_REF] Pagès | Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria[END_REF]. Quinazoline compounds had no effect on the antibiotic susceptibility of an Escherichia coli acrABdeleted strain (data not shown), demonstrating that the variation in antibiotic susceptibility is related to overexpression of efflux pump activity.

The compounds were able to reduce resistance of P. aeruginosa strains to chloramphenicol and nalidixic acid (Table 2). A similar effect was also noted with ciprofloxacin (data not shown). These results suggest that the compounds, similarly to PAN, are able to act on P. aeruginosa resistant strains and to restore, at least partially, susceptibility to two different antibiotic classes.

Discussion

In this study, 17 quinazoline derivatives were evaluated for their ability to reverse antibiotic resistance. Compounds 1187, 1188, 1189 and 1190 increased the activity of chloramphenicol against a panel of MDR Gram-negative isolates. The partial recovery of antibiotic susceptibility obtained with these compounds as well as with the EPI PAN indicates that other non-efflux pump mechanisms, such as alteration of membrane permeability, target mutation etc., are responsible in part for the total antibiotic resistance of the strains [START_REF] Pagès | Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria[END_REF]. An increase in intracellular accumulation of chloramphenicol is noted when the compounds are present during incubation of two chloramphenicol-resistant strains that overexpress the AcrAB efflux pump. In addition, the increase in intracellular concentration produced by compound 1188 in The efficiency of restoration depends on the respective affinities of the transported drugs and the compounds internal sites located inside the efflux pump; these sites have been proposed from crystallographic and modelling studies carried out with the AcrB pump [START_REF] Murakami | Multidrug efflux transporter, AcrB-the pumping mechanism[END_REF][START_REF] Pos | Drug transport mechanism of the AcrB efflux pump[END_REF]. A recent paper presented differences in the affinity constant for some antibiotics [START_REF] Nagano | Kinetic behavior of the major multidrug efflux pump AcrB of Escherichia coli[END_REF]. Consequently, as the concentration of a determined compound is increased, competition for these internal binding sites favours the compound depending on the respective affinity constant ratio, thereby promoting intrabacterial retention of the antibiotic. This effect results in increased antibiotic activity on the target.

We observed a significant increase in susceptibility to chloramphenicol and nalidixic acid, which are substrates of efflux pumps in K. pneumoniae, E. aerogenes and P. aeruginosa [START_REF] Li | Efflux-mediated drug resistance in bacteria: an update[END_REF][START_REF] Poole | Efflux pumps as antimicrobial resistance mechanisms[END_REF][START_REF] Pagès | Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria[END_REF]. This is especially important taking into account the difference in the major efflux pumps expressed in these bacteria, e.g. AcrAB-TolC and MexAB-OprM.

The difference in the restoration level obtained in the present study with the various quinazoline derivatives and the antibiotics tested may reflect: (i) a variation in the respective location of ligands inside the AcrB cavity such that binding of the compound interferes with the extrusion of the antibiotic; (ii) competition between the antibiotic and derivative for the same pump active site; (iii) the compounds differ with respect to the degree of affinity for a specific internal site; and (iv) binding of the A c c e p t e d M a n u s c r i p t 12 compound reduces the affinity of the antibiotic to a different site. The presence of different sites located inside the AcrB pump has been reported [START_REF] Yu | A periplasmic drug-binding site of the AcrB multidrug efflux pump: a crystallographic and site-directed mutagenesis study[END_REF]. With the recent model illustrating functioning of the AcrB pump [START_REF] Murakami | Multidrug efflux transporter, AcrB-the pumping mechanism[END_REF][START_REF] Pos | Drug transport mechanism of the AcrB efflux pump[END_REF], it is possible that the same amino acid residues participated at different drug binding sites located in the pump tunnel. Moreover, several recent data on the E. coli AcrB efflux mechanism indicate that some amino acid residues are involved in the transport process to a varying extent during specific steps such as binding, transport, etc. [START_REF] Yu | A periplasmic drug-binding site of the AcrB multidrug efflux pump: a crystallographic and site-directed mutagenesis study[END_REF][START_REF] Wehmeier | Site-directed mutagenesis reveals amino acid residues in the Escherichia coli RND efflux pump AcrB that confer macrolide resistance[END_REF][START_REF] Nikaido | Mechanisms of RND multidrug efflux pumps[END_REF]. This can explain the divergence observed in the competitive capacity of the compounds with drugs of different classes (chloramphenicol, quinolones, ethidium bromide) for binding sites within the pump type (AcrB, MexB) as well as the level of expressed efflux pump activity in the tested strains. Kinetic studies have reported variation in the affinity constant of various -lactams for AcrB in E. coli cells [START_REF] Nagano | Kinetic behavior of the major multidrug efflux pump AcrB of Escherichia coli[END_REF]. This flexibility of the substrate site may be directly involved in the polyselectivity of the efflux pump; a site that can be modified by site-directed mutagenesis [START_REF] Wehmeier | Site-directed mutagenesis reveals amino acid residues in the Escherichia coli RND efflux pump AcrB that confer macrolide resistance[END_REF][START_REF] Nikaido | Mechanisms of RND multidrug efflux pumps[END_REF]. The role and overlap of affinity sites may explain the discrepancy observed in the increase of intracellular concentrations of different transported molecules such as chloramphenicol, quinolones and -lactams. It is important to note that a chemosensitizer can restore a high level of intracellular antibiotic concentration, but it cannot bypass other resistance mechanisms such as target mutation or altered permeability. Moreover, some isolates may also overproduce other efflux pumps that are less susceptible to inhibitors [START_REF] Pagès | Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria[END_REF].

Among the quinazolines derivatives, the tested compounds containing a nitro functional group that decreases the electronic density of the aromatic ring are more efficient than those that are devoid of this group. Structure-activity relationship a Values are the mean of three independent determinations.

b The various molecules are used at low concentrations against the respective bacterial strains (<0.25 the determined MICs).

  family of molecules may compete with antibiotic transport within the AcrB pump[START_REF] Pagès | Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria[END_REF].

  [ 14 C]-chloramphenicol (Aventis Pharma, Romainville, France) was added to the cell suspension, yielding a final concentration of 5 M. The cell suspension was maintained at 37 C for 2 min, then 100 L of the suspension was removed and immediately filtered through GF/C filters (Whatman International Ltd., Maidstone, UK). After three washes with 4 mL of cold phosphate/LiCl 0.1 M buffer, filters were dried and the radioactivity (counts per min) was measured in a Packard scintillation counter (Perkin-Elmer Life and Analytical Sciences, Wellesley, MA). Quinazoline derivatives and phenylalanine-arginine naphthylamide (PAN) were added 10 min before [ 14 C]-chloramphenicol to perform inhibition assays.

7

 7

  A c c e p t e d M a n u s c r i p t 8 the selection of concentrations that have no effect on the growth and viability of the strain.

A

  c c e p t e d M a n u s c r i p t 11 the two resistant strains is dose-dependent. These results provide evidence that these quinazoline derivatives restore the intracellular antibiotic concentration in Gram-negative bacteria that overexpress efflux mechanisms.

  derivatives may be fruitfully undertaken to identify pharmacophoric groups involved in affinity site recognition. With the resolution of the three-dimensional structure of pump components and the recent models proposed for the pump function[START_REF] Murakami | Multidrug efflux transporter, AcrB-the pumping mechanism[END_REF][START_REF] Pos | Drug transport mechanism of the AcrB efflux pump[END_REF], development of efficient molecules that may restore the intracellular concentration of antibiotics in MDR bacterial pathogens is a promising way to circumvent efflux pump activity.

16 Fig. 1 .

 161 Fig. 1. Intrabacterial accumulation of drugs and the effect of quinazoline compounds.

1 Table 1

 11 Activity of quinazoline compounds on Enterobacter aerogenes strains a

	Edited Table 1
	Molecule Quinazoline MIC (mM) ATCC 13048 CM-64 EA27 ATCC 13048 CM-64 EA27 Chloramphenicol MIC (mg/L) 0 8 128 1024 1181 5 5 5 4 16 256 1183 10 >5 >5 4 32 512 M a n u s c r i p t	1
	1184 1180 1185 1182 1186 1174 1176 1177 1188 1189	5 5 5 10 5 10 10 10 1.25 2.5	5 5 >5 5 5 >5 >5 >5 1.25 5 A c c e p t e d 5 4 5 4 5 4 5 8 5 4 >5 4 >5 4 >5 4 1.25 4 5 2	64 16 32 32 32 32 32 64 32 32	256 128 256 256 256 256 256 512 256 256
	1187	5	5	5	1	8	64
	1193	>5 b	>5 b	>5 b	8 b	32 b	256 b
	1190	2.5	2.5	2.5	2	32	256
	1191	2.5	5	5	4	32	512
	1192	10	10	10	2	64	512
	PAN	5	5	5	≤1	≤ 16	≤ 16
	MIC, minimal inhibitory concentration; PAN, phenylalanine arginine -
	naphthylamide.

a Values are the mean of three independent determinations. b Precipitates observed.

Table 2

 2 Effects of quinazolines on the antibiotic susceptibility of Enterobacter aerogenes, Klebsiella pneumoniae and Pseudomonas aeruginosa strains a

	Molecule	MIC (mg/L)
	b	E. aerogenes

MIC, minimal inhibitory concentration; NAL, nalidixic acid; NFX, norfloxacin; CPX, ciprofloxacin; SPX, sparfloxacin; ERY, erythromycin; CHL, chloramphenicol; TIG, tigecycline; PAN, phenylalanine arginine -naphthylamide.

Acknowledgments

The authors thank Wyeth Pharmaceuticals France for the generous donation of tigecycline. Thanks also go to J.M. Bolla and A. Davin-Regli for helpful discussions.

Funding

This study was supported by the Université de la Méditerranée and Service de Santé des Armées (livre rouge operation23e). Support was also obtained through COST Action BM0701 (ATENS).

Competing interests

None declared.

Ethical approval

Not required. Edited Figure 1


James Carroll

Lorraine A Draper

Paula M O'connor

Aidan Coffey

Colin Hill

R Paul Ross

Paul D Cotter

Jim O'mahony
email: jim.omahony@cit.ie

Comparison of the activities of the lantibiotics nisin and lacticin 3147 against clinically significant mycobacteria

Keywords:

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

ABSTRACT

The aim of this study was to use the microtitre alamarBlue assay to investigate and compare the antimycobacterial potential of the lantibiotics nisin and lacticin 3147 against a representative cohort of clinically significant mycobacteria, i.e.

Mycobacterium tuberculosis H37Ra, Mycobacterium avium subsp. paratuberculosis (MAP) ATCC 19698 and Mycobacterium kansasii CIT11/06. Lacticin 3147 displayed potent activity against all strains of mycobacteria, with MIC 90 values (lowest concentration of lantibiotic that prevented growth of >90% of the bacterial population) of 60 mg/L and 15 mg/L for M. kansasii and MAP, respectively. Lacticin 3147 was particularly effective against M. tuberculosis H37Ra, with an MIC 90 value of 7.5 mg/L. Nisin, although inhibitory, was generally less potent against all strains of mycobacteria, with MIC 90 values of 60 mg/L for M. kansasii and >60 mg/L for MAP and M. tuberculosis H37Ra. Thus, lacticin 3147 is a potent antimycobacterial peptide that shows superior activity compared with nisin at physiological pH.

Introduction

The extent and diversity of mycobacterial disease is a global problem, impacting significantly both on human and animal health. Mycobacterium tuberculosis is a Gram-positive pathogen that is the main aetiological agent of tuberculosis (TB), and recent statistics from the World Health Organization (WHO) estimate that there were 9.27 million incident cases of TB in 2007 [START_REF]Global tuberculosis control-epidemiology, strategy, financing[END_REF].

Furthermore, with an estimated 500 000 multidrug-resistant TB (MDR-TB) cases and at least one case of extensively-drug-resistant TB (XDR-TB) reported in 55 countries and territories by the end of 2008 [START_REF]Global tuberculosis control-epidemiology, strategy, financing[END_REF], the identification and development of novel antimicrobial compounds is now more important than ever. However, with the exception of rifabutin and rifapentine, no novel drugs have been marketed for TB in the 40 years following the release of rifampicin [START_REF] Tomioka | Development of antituberculous drugs: current status and future prospects[END_REF]. An additional worry is the increasing number of pathogenic mycobacteria other than M. tuberculosis, which are being implicated as causative agents of disease [START_REF] Field | Lung disease due to the more common nontuberculous mycobacteria[END_REF], especially in acquired immune deficiency syndrome (AIDS) patients. One such species, Mycobacterium avium subsp. paratuberculosis (MAP), the aetiological agent of Johne's disease in animals, has caused significant controversy, with reports suggesting its association with Crohn's disease in humans [START_REF] Hermon-Taylor | Causation of Crohn's disease by Mycobacterium avium subspecies paratuberculosis[END_REF][START_REF] Scanu | Mycobacterium avium subspecies paratuberculosis infection in cases of irritable bowel syndrome and comparison with Crohn's disease and Johne's disease: common neural and immune pathogenicities[END_REF][START_REF] Nacy | Mycobacterium avium paratuberculosis: infrequent human pathogen or public health threat? August 2008[END_REF]. Another mycobacterium, Mycobacterium kansasii, is responsible for serious pulmonary infections and some non-life threatening infections such as septic arthritis [START_REF] Williams | Mycobacterium kansasii causing septic arthritis and osteomyelitis in a child[END_REF].

In recent years, an enhanced appreciation of the efficacy of the antimicrobials known as lantibiotics has resulted in their being the focus of much attention as a possible A c c e p t e d M a n u s c r i p t 4 alternative to current antibiotic regimens [START_REF] Cotter | Bacterial lantibiotics: strategies to improve therapeutic potential[END_REF][9][START_REF] Russell | The bacteriocins of ruminal bacteria and their potential as an alternative to antibiotics[END_REF][START_REF] Gillor | Genetically engineered bacteriocins and their potential as the next generation of antimicrobials[END_REF][START_REF] Asaduzzaman | Lantibiotics: diverse activities and unique modes of action[END_REF][START_REF] Willey | Lantibiotics: peptides of diverse structure and function[END_REF][START_REF] Bierbaum | Lantibiotics: mode of action, biosynthesis and bioengineering[END_REF]. Lantibiotics are gene-encoded, posttranslationally-modified antimicrobial peptides [START_REF] Cotter | Bacterial lantibiotics: strategies to improve therapeutic potential[END_REF] that are distinguished by the unusual lanthionine and/or -methyllanthionine residues, dehydrated serines and/or threonines [START_REF] Cotter | Bacterial lantibiotics: strategies to improve therapeutic potential[END_REF]. To date, more than 50 different lantibiotics have been identified with different structures and modes of action [START_REF] Willey | Lantibiotics: peptides of diverse structure and function[END_REF], and recent comprehensive reviews on lantibiotics have highlighted their potential therapeutic applications [START_REF] Asaduzzaman | Lantibiotics: diverse activities and unique modes of action[END_REF][START_REF] Willey | Lantibiotics: peptides of diverse structure and function[END_REF][START_REF] Bierbaum | Lantibiotics: mode of action, biosynthesis and bioengineering[END_REF].

Nisin A and lacticin 3147 are two of the most extensively characterised lantibiotics.

Nisin is the prototypical lantibiotic and has been approved by the US Food and Drug Administration (FDA) for use in food and veterinary products [START_REF] Delves-Broughton | Nisin and its application as a food preservative[END_REF]. Lacticin 3147 differs from nisin by virtue of requiring two peptides for optimal activity, i.e. Ltn and Ltn (formerly LtnA1 and LtnA2) [START_REF] Morgan | Sequential actions of the two component peptides of the lantibiotic lacticin 3147 explain its antimicrobial activity at nanomolar concentrations[END_REF]. Notably, both nisin and lacticin 3147 are active at nanomolar concentrations [START_REF] Morgan | Sequential actions of the two component peptides of the lantibiotic lacticin 3147 explain its antimicrobial activity at nanomolar concentrations[END_REF] and can inhibit antibiotic-resistant bacteria [START_REF] Piper | A comparison of the activities of lacticin 3147 and nisin against drug-resistant Staphylococcus aureus and Enterococcus species[END_REF].

Moreover, both antimicrobials possess more than one mechanism of action in that they inhibit peptidoglycan synthesis as well as forming pores in the membrane of target cells. Both activities are facilitated by binding to the peptidoglycan precursor, lipid II [START_REF] Wiedemann | Specific binding of nisin to peptidoglycan precursor lipid II combines pore 16 formation and inhibition of cell wall biosynthesis for potent antibiotic activity[END_REF][START_REF] Breukink | Use of the cell wall precursor lipid II by a pore-forming peptide antibiotic[END_REF][START_REF] Brötz | Role of lipid-bound peptidoglycan precursors in the formation of pores by nisin, epidermin and other lantibiotics[END_REF][START_REF] Wiedemann | The mode of action of the lantibiotic lacticin 3147-a complex mechanism involving specific interaction of two peptides and the cell wall precursor lipid II[END_REF], which is also a target for some conventional antimycobacterials such as rifampicin that require transportation across the plasma membrane before they can be effective [START_REF] Hartmann | Molecular mechanism of action of the antibiotic rifampicin[END_REF]. It is notable that the structure of mycobacterial lipid II differs from all other structures in bacteria investigated to date. This is due to an alternative synthesis pathway as well as modifications both on the N-acetylmuramic acid and peptide side chain components [START_REF] Mahapatra | Mycobacterial lipid II is composed of a complex mixture of modified muramyl and peptide moieties linked to decaprenyl phosphate[END_REF]. Peptidoglycan synthesis in mycobacteria is depicted in Fig. 1.

A c c e p t e d M a n u s c r i p t 5 Whilst nisin has been found to be active against non-pathogenic Mycobacterium smegmatis and Mycobacterium bovis strains [START_REF] Montville | Nisin A depletes intracellular ATP and acts in a bactericidal manner against Mycobacterium smegmatis[END_REF], neither nisin nor lacticin 3147 have been comprehensively assessed with respect to their antimicrobial activity against clinically significant human and animal mycobacterial pathogens.

Materials and methods

Mycobacterium strains and culture conditions

Mycobacterium kansasii CIT11/06 (10 days) and M. tuberculosis H37Ra (11 days) were routinely grown at 37 C in Middlebrook 7H9GC broth (MB) (Sigma-Aldrich, Arklow, Co. Wicklow, Ireland) supplemented with glycerol (0.2%) and 10% oleic acid-albumin-dextrose-catalase (OADC) (Unitech, Dublin, Ireland). MAP ATCC 19698, also grown in MB containing OADC, was supplemented with 0.2% mycobactin J (Synbiotics Europe, Lyon, France) and was incubated at 37 C for 8-12 weeks. All strains were stocked in 40% glycerol and stored at -80 C.

Preparation of pure lacticin 3147 and nisin A

The lantibiotics were purified as described previously [START_REF] Piper | A comparison of the activities of lacticin 3147 and nisin against drug-resistant Staphylococcus aureus and Enterococcus species[END_REF][START_REF] Field | The generation of nisin variants with enhanced activity against specific Gram-positive pathogens[END_REF]. In brief, peptides were concentrated via centrifugation and rotary evaporation before being applied to a Phenomenex C12 reversed-phase high-performance liquid chromatography (RP-HPLC) column (Phenomenex, Cheshire, UK) equilibrated with 25% propan-2-ol and 0.1% trifluoroacetic acid (TFA). The column was subsequently developed in a A c c e p t e d M a n u s c r i p t 6 gradient of 30% propan-2-ol containing 0.1% TFA to 60% propan-2-ol containing 0.1% TFA at a flow rate of 1.2 mL/min.

Preparation of mycobacterial cells

Owing to the slow-growing nature of pathogenic mycobacteria, standard agar and broth minimum inhibitory concentration (MIC) assay methods could not be performed routinely. Thus, the reliable and established microtitre alamarBlue assay was employed [START_REF] Bastian | Kanamycin susceptibility testing of Mycobacterium tuberculosis using Mycobacterium Growth Indicator Tube and a colorimetric method[END_REF][START_REF] Franzblau | Rapid, low-technology MIC determination with clinical Mycobacterium tuberculosis isolates by using the microplate alamarBlue assay[END_REF][START_REF] Carroll | Optimisation of a rapid viability assay for Mycobacterium avium paratuberculosis (MAP) using alamarBlue[END_REF]. Isolates of M. kansasii, MAP and M. tuberculosis were grown in MB as described above. At log phase, a 10 mL culture of each mycobacterial strain was centrifuged at 15 000 rpm for 20 min using a benchtop centrifuge. Following removal of the supernatant, the pellet was washed in fresh MB and re-suspended in 10 mL of fresh supplemented MB. The turbidity was adjusted to match a McFarland standard no. 1 (3  10 8 colony-forming units/mL) with MB. A further 1:20 dilution of the culture was then performed in MB to generate a sufficient volume of culture.

Determination of minimum inhibitory concentrations against clinical isolates using the microtitre alamarBlue assay

Sterile deionised water (300 L) was added to all outer-perimeter wells of a 96-well plate to minimise evaporation of the medium in the test wells during incubation. In the same 96-well plate, a serial 1:2 dilution of each lantibiotic (lacticin 3147 contained equimolar concentrations of both Ltn and Ltn) was then performed in MB (100 L). Final concentrations ranged from 0.11 mg/L to 60 mg/L and each dilution series was prepared in triplicate. Positive controls were prepared by adding Negative controls contained no bacterial cells. Positive and negative controls were also prepared in triplicate. Subsequently, an equal volume of the mycobacterial strain to be assessed was transferred to each well under investigation. The plates were sealed with Parafilm TM and incubated at 37 C. Following incubation for 3 days, 7 days and 11 days for M. kansasii, M. tuberculosis and MAP, respectively, 20 L (10% of the final volume in the well) of a fresh alamarBlue reagent (AbD Serotec, Oxford, UK) was added to each well and the plates were re-incubated for 24 h, after which time absorbance at 570 nm and 600 nm was determined. Subsequent percent reduction values based on alamarBlue conversion were determined using the appropriate formula [START_REF]alamarBlue ® technical datasheet[END_REF] and the relative growth in each well was compared with that of the positive control. The MIC 90 was defined as the lowest concentration of lantibiotic that prevented the growth of >90% of the bacterial population relative to the positive control.

Results

AlamarBlue-based minimum inhibitory concentration determination

AlamarBlue is a redox indicator that yields a colorimetric change in a response to metabolic activity [START_REF] Carroll | Optimisation of a rapid viability assay for Mycobacterium avium paratuberculosis (MAP) using alamarBlue[END_REF]. Relative inhibition of mycobacteria by nisin and lacticin 3147 was assessed by quantifying the absorbance (570 nm and 600 nm) of all wells to facilitate the calculation of dye reduction values (%) using the appropriate formula [START_REF]alamarBlue ® technical datasheet[END_REF]. Wells that remained blue (i.e. low dye reduction values) were indicative of inhibition of the target strain, whilst wells that turned pink (high dye reduction values)

A c c e p t e d M a n u s c r i p t 8 were indicative of growth. From these calculated dye reduction values, the relative growth (%) and corresponding inhibition values were obtained, taking the value of the positive control in each case as 100%. From these values it was possible to determine the MIC 90 value. Overall, the relative growth of all test strains varied from 1.2% to 71.6% under the experimental conditions examined. To reflect physiological pH, assays were carried out at pH 7.

MIC 90 values for nisin and lacticin 3147 against Mycobacterium tuberculosis

H37Ra

Mycobacterium tuberculosis H37Ra was the most sensitive strain to lacticin 3147, with an MIC 90 value of 7.5 mg/L (Table 1; Fig. 2). At this concentration, the relative growth of M. tuberculosis was inhibited by 91%. Increasing the lacticin 3147 concentration further resulted in even greater inhibition and culminated in 98.8% inhibition in the presence of 60 mg/L lacticin 3147 (the highest concentration employed). Interestingly, lacticin 3147 also exhibited activity at the lowest concentration employed (0.11 mg/L), where >50.3% inhibition was observed.

With respect to nisin, the highest concentration of the lantibiotic used (60 mg/L) failed to inhibit the pathogen by 90% and thus the MIC 90 value was recorded as >60 mg/L. The assays performed did allow some insight into the sensitivity of the strain to nisin in that it was shown that nisin inhibited 76.3% of M. tuberculosis culture at 60 mg/L nisin. At 30 mg/L nisin, 68.2% of the M. tuberculosis growth was inhibited and subsequent dilutions of nisin established that it is still active at concentrations as low as 0.11 mg/L, as demonstrated by 50.9% inhibition at this concentration (Fig. 2).

A c c e p t e d

M a n u s c r i p t 9

MIC 90 values for nisin and lacticin 3147 against Mycobacterium kansasii CIT11/06

Of the strains tested, M. kansasii CIT11/06 was the least susceptible to lacticin 3147, with an MIC 90 value of 60 mg/L (Table 1; Fig. 3). Relative growth inhibition was 93% at this concentration (Fig. 3). Below 15 mg/L, the potency of lacticin 3147 became significantly decreased. However, even at the lowest concentration of lacticin 3147 employed (0.11 mg/L), activity was still apparent as represented by a 29.2% inhibition of M. kansasii (Fig. 3).

In comparison, nisin was more active against M. kansasii CIT11/06 than the other strains tested, with an MIC 90 value of 60 mg/L. At this concentration, 94.5% inhibition of M. kansasii growth was noted. This dropped to 67.5% inhibition at 30 mg/L nisin and, as expected, even further for the lower concentrations. However, even at the lowest concentration of nisin tested (0.11 mg/L), inhibitory activity was still detected (30.3% inhibition) (Fig. 3).

MIC 90 values for nisin and lacticin 3147 against Mycobacterium avium paratuberculosis ATCC 19698

For lacticin 3147, the MIC 90 for MAP ATCC 19698 was 15 mg/L (90.6% reduction) (Table 1; Fig. 4). Moreover, 95.2% and 94% inhibition of the MAP population was recorded at concentrations of 60 mg/L and 30 mg/L, respectively, whilst strong inhibition (86%) was also observed when 7.5 mg/L lacticin 3147 was utilised (Fig. 4). Although nisin showed quite good efficacy against MAP, 60 mg/L nisin failed to bring about 90% inhibition, with inhibition peaking at 81% (Fig. 4). A decrease in efficacy was seen with lower concentrations of nisin, with moderate activity still apparent at very low concentrations (0.46-0.11 mg/L) (Fig. 4).

Comparison of the relative antimycobacterial activities of lacticin 3147 and nisin

Under these experimental conditions, lacticin 3147 consistently showed superior activity at higher concentrations (Table 1). Mycobacterium tuberculosis was the most susceptible species of mycobacteria, particularly to lacticin, which was 8 times more potent than nisin (Fig. 2). Lacticin 3147 brought about as much as 38% greater inhibition at equivalent concentrations compared with nisin (7.5 mg/L). Whilst this difference in relative growth remained relatively constant between the concentrations of 15-7.5 mg/L, below 0.93 mg/L both nisin and lacticin 3147 had an equivalent inhibitory effect against M. tuberculosis.

Lacticin 3147 also exhibited superior potency against M. kansasii (Fig. 3), with 31% less relative growth in the presence of lacticin 3147 compared with nisin (15 mg/L).

Although the MIC 90 values for both lantibiotics were the same, the general trend of inhibition indicated that lacticin 3147 was more potent. However, at concentrations <1.8 mg/L, the activity of both lantibiotics was comparable.

A c c e p t e d M a n u s c r i p t 11 Lacticin 3147 also displayed strong inhibitory activity against MAP, with an MIC 90 value 4-fold lower than that of nisin (15 mg/L and <60 mg/L, respectively) (Fig. 4).

As with the other two strains, both lantibiotics had similar inhibitory capacity at lower concentrations (e.g. 0.11 mg/L). The greatest difference in percent relative growth between both lantibiotics at equivalent concentrations was seen between 15 mg/L and 3.75 mg/L. This relative difference remained almost constant over these three concentrations (23.6-24.6% relative growth).

Discussion

Antimicrobial peptides are rapidly becoming a realistic alternative therapy, with many peptides showing potent activity against pathogens that has led to their evaluation in clinical trials [START_REF] Zaiou | Multifunctional antimicrobial peptides: therapeutic targets in several human diseases[END_REF]. Although lantibiotics are traditionally associated with food applications [START_REF] Cotter | Bacteriocins: developing innate immunity for food[END_REF], it is evident from the literature that they possess notable levels of activity against clinical pathogens [START_REF] Piper | A comparison of the activities of lacticin 3147 and nisin against drug-resistant Staphylococcus aureus and Enterococcus species[END_REF][START_REF] Brumfitt | Nisin, alone and combined with peptidoglycan-modulating antibiotics: activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci[END_REF][START_REF] Davies | The use of the bacteriocin, nisin, as a preservative in ricotta-type cheeses to control the food-borne pathogen Listeria monocytogenes[END_REF][START_REF] Severina | Antibacterial efficacy of nisin against multidrug-resistant Gram-positive pathogens[END_REF][START_REF] Rea | Antimicrobial activity of lacticin 3147 against clinical Clostridium difficile strains[END_REF][START_REF] Galvin | Lacticin 3147 displays activity in buffer against Gram-positive bacterial pathogens which appear insensitive in standard plate assays[END_REF]. However, this study is notable in that it is the first occasion upon which the efficacy of lacticin 3147 against a Mycobacterium sp. has been tested and compared with the activity of nisin. It is also significant that the mycobacteria chosen for this study (which are implicated both in human and animal disease) showed considerable susceptibility to both agents. This study also establishes lipid II as a promising antimycobacterial target and confirms that binding of lantibiotics to the mycobacterial lipid II is not compromised by the extensively modified lipid II structure in mycobacteria [START_REF] Mahapatra | Mycobacterial lipid II is composed of a complex mixture of modified muramyl and peptide moieties linked to decaprenyl phosphate[END_REF].

Of the two lantibiotics, lacticin 3147 demonstrated greater potential as a therapeutic agent. This can be attributed to its activity at physiological pH 7, which contrasts with A c c e p t e d M a n u s c r i p t 12 that of nisin that is poorly soluble, and thus less active, at this pH [START_REF] Cotter | Bacterial lantibiotics: strategies to improve therapeutic potential[END_REF][START_REF] Galvin | Lacticin 3147 displays activity in buffer against Gram-positive bacterial pathogens which appear insensitive in standard plate assays[END_REF]. The lower activity of nisin at physiological pH has reportedly also been an issue with respect to its use in the treatment of mastitis-causing pathogens [START_REF] Cotter | Bacterial lantibiotics: strategies to improve therapeutic potential[END_REF]. From the data presented here, it is apparent that lacticin 3147 inhibits mycobacteria at concentrations that are comparable with those of antibiotics currently employed against these pathogens [START_REF] Wanger | Testing of Mycobacterium tuberculosis susceptibility to ethambutol, isoniazid, rifampin, and streptomycin by using Etest[END_REF][START_REF] Heifets | Ethambutol MICs and MBCs for Mycobacterium avium complex and Mycobacterium tuberculosis[END_REF]. These values are in some cases superior to those of the first-line antimycobacterial drug isoniazid that had MIC values of 4-16 mg/L as determined by Wanger and Mills [START_REF] Wanger | Testing of Mycobacterium tuberculosis susceptibility to ethambutol, isoniazid, rifampin, and streptomycin by using Etest[END_REF].

Whilst the in vitro efficacy of lacticin 3147 against mycobacteria is encouraging, stability, delivery and in vivo efficacy studies will all need to proceed before the true potential of the lantibiotic as a clinical antimycobacterial can be assessed. The unique ability of some mycobacteria to reside in the phagosomal site within pulmonary macrophages makes their eradication extremely difficult. Moreover, the delivery of antimycobacterial agents that traverse the plasma membrane and maintain stability and activity is rare. However, recent studies have demonstrated that administration of bacteriocins as a complex with liposomes provides a successful mechanism of delivery and inhibition, thus prolonging survival time of mice in an acute TB model [START_REF] Sosunov | Antimycobacterial activity of bacteriocins and their complexes with liposomes[END_REF]. In this context, lantibiotics undoubtedly possess sufficient potential to merit further investigation.

  A c c e p t e d M a n u s c r i p t 7 100 L of the appropriate mycobacterial strain to 100 L of lantibiotic-free broth.

A

  c c e p t e d M a n u s c r i p t 10 At the lowest concentration employed (0.11 mg/L), lacticin 3147 successfully inhibited >50% of the MAP population.

19 Fig. 1 .

 191 Fig. 1. Schematic cross-section of the mycobacterial cell envelope. The model

Fig. 2 .

 2 Fig. 2. Relative growth of Mycobacterium tuberculosis H37Ra in the presence of

Fig. 3 .

 3 Fig. 3. Relative growth of Mycobacterium kansasii CIT11/06 in the presence of nisin

Fig. 4 .

 4 Fig. 4. Relative growth of Mycobacterium avium subsp. paratuberculosis ATCC

1 1

 1 = H,COCH 3 (N-acetyl) or COCH (N-glycol)

Table 1

 1 MIC 90 values of lacticin 3147 and nisin against pathogenic mycobacteria MIC 90 , lowest concentration of lantibiotic that prevented growth of >90% of the bacterial population; MAP, Mycobacterium avium subsp. paratuberculosis.

	Edited
	Lantibiotic	MIC 90 (mg/L)
		M. tuberculosis H37Ra M. kansasii CIT11/06 MAP ATCC 19698
	Lacticin 3147 7.5 ( 0.36)	60 ( 0.75)	15 ( 0.30)
	Nisin	>60 ( 0.18)	60 ( 0.13)	>60 ( 2.02)

Table 1

 1

Acknowledgments

The authors would like to thank Cork University Hospital (Cork, Ireland) for providing the Mycobacterium kansasii strain, Dr Joe Keane (St James' Hospital, Dublin,

Funding

This research was funded by a Strand I research grant from the Technological Strand Research Program awarded through the Institute of Technology Ireland initiative. JO and PDC are recipients of a Department of Agriculture grant (MAPSAFE).

Competing interests

None declared.

Ethical approval

Not required.

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


S Kocher

Dr Bernhard Resch
email: bernhard.resch@medunigraz.at

Linezolid treatment of nosocomial bacterial infection with multiresistant Gram-positive pathogens in preterm infantsa systematic review

Abstract

Linezolid is an antibiotic of the oxazolidinone class that has bacteriostatic and bactericidal activity against a broad range of Gram-positive bacteria, including multiresistant pathogens. Due to increasing resistance of Gram-positive pathogens to traditional antibiotics like vancomycin the oxazolidinones where introduced into therapy. Aim of this review was to summarize actual data on pharmacokinetics, safety and clinical use of linezolid in preterm infants. MEDLINE and EMBASE were searched using the term "linezolid" combined with "newborn", "neonate", "preterm" and "premature" between 1988 and 2008. Studies reporting on a population including preterm infants and other age-groups and case reports on preterm infants only were acceptable for analysis. Five studies and eight case reports were identified evaluating linezolid in preterm infants. A dosage regimen of 10 mg/kg body weight given either orally or intravenously every 8 hours in infants aged one week or more and the same dose given every 12 hours in infants younger than one week was shown to be safe and effective with mean treatment duration of 10 to 28 days.

In summary linezolid has shown to be a safe and effective alternative to vancomycin in the treatment of infections with multiresistant Gram-positive pathogens in preterm infants.

Key words: linezolid, preterm infant, gram-positive bacteria, nosocomial infection A c c e p t e d M a n u s c r i p t

Introduction

Gram-positive pathogens are the most common and troublesome causes of nosocomial infections in neonatal intensive care units particularly coagulasenegative staphylococci (CONS) and enterococci 1,2 . Those pathogens accumulate with indwelling catheters and focal complications associated with prematurity and can be the cause for early-and more over late-onset sepsis [START_REF] Stoll | Early-onset sepsis in very low birth weight neonates: A report from the National Institute of Child Health and Human Development Neonatal Research Network[END_REF][START_REF] Stoll | Late-onset sepsis in very low birth weight neonates: A report from the National Institute of Child Health and Human Development Neonatal Research Network[END_REF] .

Due to increasing resistance of these pathogens to traditional antibiotic therapy like ampicillin, aminoglycosides and cefotaxime, glycopeptides like vancomycin are often required for adequate therapy. Unfortunately it is assumed that the potential overuse of vancomycin might be dangerous (i.e., need for parenteral therapy with the risk of phlebitis or other injection reactions, unnecessary use of a nephrotoxic drug, increased risk of antibiotic resistance and possible colonization with multiply resistant organisms, fungi, or both) [START_REF] Stoll | Late-onset sepsis in very low birth weight neonates: A report from the National Institute of Child Health and Human Development Neonatal Research Network[END_REF] Therefore linezolid, a member of the oxazolidinones, a new class of antibiotics with a completely novel mechanism of action [START_REF] Slee | Oxazolidinones, a new class of synthetic antibacterial agents: in vitro and in vivo activities of DuP 105 and DuP 721[END_REF] [START_REF] Wise | The in vitro activity of linezolid (U-100766) and dentative beakpoints[END_REF][START_REF] Diekema | Oxazolidinone antibiotics[END_REF][START_REF] Moellering | Linezolid: the first oxazolidinone antimicrobial[END_REF][START_REF] Stevens | A review of linezolid: the first oxazolidinone antibiotic[END_REF] .

Linezolid is also active against anaerobes, atypical microbes such as A c c e p t e d M a n u s c r i p t 4 Chlamydia and Mycoplasma spp., some rapidly growing mycobacteria and selected Gram-negative bacilli [START_REF] Breha-Millet | Activity of linezolid against anaerobic bacteria[END_REF][START_REF] Wallace Rj | Activities of linezolid against rapidly growing mycobacteria[END_REF]12 . Linezolid or N-([S]-[3-fluoro-4morpholinophenyl]-2-oxo-5-oxazolidinyl] methyl) acetamide is a totally synthetic antimicrobial agent structurally unrelated to other agents with an unique mechanism of action [START_REF] Wise | The in vitro activity of linezolid (U-100766) and dentative beakpoints[END_REF][START_REF] Diekema | Oxazolidinone antibiotics[END_REF] . Thus, cross-resistance with other protein synthesis inhibitors such as aminoglycosides and macrolides is generally avoided [START_REF] Kloss | Resitance mutations in 23S rRNA identify the site of action of the protein synthesis inhibitor linezolid in the ribosomal peptidyltransferase center[END_REF] .

Moreover the development of resistance to linezolid seems difficult to achieve, requiring specific 23S recombinant RNA mutations [START_REF] Tsiodras | Linezolid resistance in a clinical isolate of Staphylococcus aureus[END_REF] .

In the U.S. linezolid is approved for paediatric patients since December 2002.

In Austria as in many other European countries its use in paediatric patients is off-label. Aim of this systematic review was to summarize actual data on pharmacokinetics and safety, as well as to outline experiences in clinical use of linezolid in preterm infants.

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

Methods

Literature sources and search terms

MEDLINE and EMBASE were searched systematically to identify all papers published in the English and German literature regarding the use of linezolid in preterm infants, including the period from January 1988 to June 2009. This time window was chosen due to the fact that all identified papers about the class of oxazolidinones and linezolid were published after 1987. For the search the following keywords were utilized both individually and in different combinations: linezolid, newborn, neonate, preterm and premature. Cross-referencing of important articles was also applied.

Study selection

Original observational or interventional population or hospital-based studies involving preterm infants < 37 weeks of gestational age with severe infections with multiresistant Gram-positive infections being treated with linezolid were included. In addition, only studies with well-defined participants' characteristics and number of subjects treated with linezolid were included in the analysis.

Data abstraction

One author extracted and screened potentially relevant articles to determine whether they met the inclusion criteria. Then data was abstracted from each included article and reviewed cross-referencing with the co-authors for additional potentially relevant articles. Abstract discrepancies were resolved by repeated review and discussion.

Results

A total of five pharmacological and/or clinical studies and eight case reports were identified. Data of pharmacological and clinical studies are summarized in table 1.

Pharmacologic properties in preterm infants

In the manufacturer's prescribing information [START_REF] Pharmacia | Zyvox: linezolid injection, linezolid tablets, linezolid for oral suspension package insert[END_REF] the pharmacokinetic properties of single dose intravenous linezolid in paediatric patients are reported. Results are based on studies of linezolid 10 mg/kg in paediatric patients aged <7 days (gestational age <34 weeks [n=9], gestational age ≥34 weeks [n=10]), 7-28 days (n=10), >28 days to 3 <3months (n=59), and 3 month to 11 years (n=59), and linezolid 600 mg or 10 mg/kg to a maximum to 600 mg in adolescent volunteers and patients aged 12-17 years (n=36) [START_REF] Pharmacia | Zyvox: linezolid injection, linezolid tablets, linezolid for oral suspension package insert[END_REF] . The pharmacokinetics in these age groups are compared with those in 29 adult subjects receiving a single intravenous infusion of linezolid (dose normalized to 600 mg) [START_REF] Pharmacia | Zyvox: linezolid injection, linezolid tablets, linezolid for oral suspension package insert[END_REF] . The studies showed that values including pharmacokinetic parameters (area under the plasma concentration-time curve [AUC], systemic clearance [CL] and elimination half-life [t½]) of linezolid change with increasing age (figure 1) [START_REF] Pharmacia | Zyvox: linezolid injection, linezolid tablets, linezolid for oral suspension package insert[END_REF] .

Preterm neonatal patients aged <1 week demonstrated a higher mean AUC than the other age groups; however, there was no difference at the age of 7 days.

In a similar manner, mean clearance for infants <7 days postnatal age is substantially less than in older infants and the CL of linezolid increases rapidly during the first week after birth in both preterm and full-term neonates [START_REF] Jungbluth | Impact of gestational and postnatal age on linezolid disposition in neonates and young infants [abstract no. 2712[END_REF] . Among preterm infants <7 days old the clearance of linezolid is lowest; clearance rates A c c e p t e d M a n u s c r i p t 8 are similar to those observed in adult patients. However, the clearance of linezolid increases markedly in the first week of life, regardless of gestational age, such that infants >7 days old have clearance values that are ≈3-fold greater than those of adults (figure 1). The age-dependent differences in the CL of linezolid may be partially the result of differences with age in activity of non-CYP isoforms (e.g. amidases, peptidases and peroxidases), although these enzymes have not specifically been identified as playing a role in linezolid metabolism [START_REF] Kearns | Single dose pharmacokinetics of linezolid in infants and children[END_REF][START_REF] Leeder | Pharmacogentetics in pediatrics: implications for practice[END_REF] . In contrast to AUC, CL and t 1/2 the age of paediatric patients does not affect the C max or steady-state volume of distribution (V SS ) of linezolid [START_REF] Pharmacia | Zyvox: linezolid injection, linezolid tablets, linezolid for oral suspension package insert[END_REF] . [START_REF] Kearns | Impact of ontogeny on linezolid disposition in neonates and infants. Pharmacokinetics and Drug Disposition[END_REF] open label, single-dose evaluation of linezolid pharmacokinetics and tolerance at a single dose level (10 mg/kg). Single-dose linezolid was well tolerated in neonates and young infants in this study. There were no clinically significant adverse events or clinically significant changes in vital signs or physical findings associated with the study drug or experimental procedures.

Kearns et al

The pharmacokinetic parameters of the study are comparable with data reported above [START_REF] Pharmacia | Zyvox: linezolid injection, linezolid tablets, linezolid for oral suspension package insert[END_REF][START_REF] Kearns | Single dose pharmacokinetics of linezolid in infants and children[END_REF] .

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

Recommended dosages and duration of treatment in preterm infants

The recommended dosage of linezolid in most indications is 10 mg/kg every 8 hours in children aged 0-11 years and 600 mg every 12 hours in adolescents aged ≥12 years [START_REF] Pharmacia | Zyvox: linezolid injection, linezolid tablets, linezolid for oral suspension package insert[END_REF] ≤4mg/L for susceptibility and ≥16mg/L for resistance [START_REF] Biedenbach | Disk diffusion test interpretative criteria and quality control recommendations for testing linezolid (U-100766) abd eperezolid (U-100592) with commercially prepared reagens[END_REF] .

No dosage changes are required when switching from intravenous to oral formula of linezolid. The recommended durations of treatment are 10-14 days in patients with skin and skin-structure infections or pneumonia and 14-28 days in those with vancomycin-resistant E. faecium infections [START_REF] Pharmacia | Zyvox: linezolid injection, linezolid tablets, linezolid for oral suspension package insert[END_REF] .

Side effectstolerability in preterm infants

Kaplan et al [START_REF] Kaplan | Linezolid versus vancomycin for treatment of resistant Gram-positive infections in children[END_REF] assessed the clinical efficacy and safety of linezolid vs.

vancomycin in 316 hospitalized children including 34 preterm infants infected with nosocomial multiresistant Gram-positive bacteria. They were randomized 2:1 to receive either linezolid iv 10mg/kg every 8 h or vancomycin iv 10 to 15

A c c e p t e d M a n u s c r i p t 10 mg/kg every 6 to 24 h at least for 3 days followed by oral linezolid or vancomycin and then by an appropriate oral agent. Treatment duration was 10 to 28 days. The overall clinical cure rate was 89% compared to 85% (p=0.31).

Pathogen eradication rates were high for both linezolid and vancomycin regarding methicillin susceptible S. aureus (95% vs. 83%, p=0,87). Linezolid treated patients required significantly fewer days of iv therapy compared with vancomycin treated patients (8.0 ± 4.8 vs. 10.9 ± 5.8, p<0.001). In addition significantly fewer linezolid-treated patients had drug-related adverse events compared to vancomycin-treated patients (19% vs. 34%, p=0.003) including rash, moniliasis, red man syndrome, pruritus and fever (see Table 2) [START_REF] Kaplan | Linezolid versus vancomycin for treatment of resistant Gram-positive infections in children[END_REF] .

Hematologic effects were uncommon and similar between treatment groups.

Details of the 34 preterm infants of the above mentioned study were reported by

Deville et al [START_REF] Deville | Linezolid versus vancomycin for treatment of resistant Grampositive infections in neonates[END_REF] focussing on the results of a total of 63 newborns. Twenty-six preterm infants <34 weeks were in the linezolid group and compared to eight in the vancomycin group. The clinical cure rate was 84% compared to 77% (p=0,553).

Pathogen eradication rates comparing both groups were 67% vs. 60% (p=0.850) for Meissner et al [START_REF] Meissner | Hematologic effects of linezolid in young children[END_REF] evaluated the occurrence of hematologic effects of linezolid compared to vancomycin in the same study group [START_REF] Deville | Linezolid versus vancomycin for treatment of resistant Grampositive infections in neonates[END_REF] . Overall, no significant differences were noted in drug-related hematologic events, such as thrombocytopenia (linezolid, 1.9% vs. vancomycin, 0%; P = 0.170), anaemia (linezolid, 1.4% vs. vancomycin, 1.0%; P = 0.771) or neutropenia (linezolid, 0% vs.

S.aureus

vancomycin, 0%). No clinically relevant changes in reticulocyte index or iron studies were noted between treatment groups.

Case reports

In our literature review we found eight case reports on a total of 14 preterm infants successfully treated with linezolid. In all cases linezolid was applied after glycopeptide treatment failures with known or suspected glycopeptide-resistant Gram-positive bacteria. Seven patients suffered from ventriculitis or an infection of the cerebral spinal fluid (CSF). As linezolid has previously been shown to have excellent penetration into the CSF in adults [START_REF] Hachem | Successful treatment of vancomycin-resistant Enterococcus meningitis with linezolid[END_REF] , it also achieved good concentrations in CSF in those preterm infants [START_REF] Graham | Linezolid treatment of vancomycin-resistant Enterococcus faecium ventriculitis[END_REF][START_REF] Kumar | Treatment of vancomycin-resistant Enterococcus faecium ventriculitis in a neonate[END_REF][START_REF] Langgartner | Linezolid for treatment of catheterrelated cerebrospinal fluid infections in preterm infants[END_REF] . Another two patients suffered from endocarditis [START_REF] Ang | Vancomycin resistant Enterococcus faecium endocarditis in a premature infant successfully treated with linezolid[END_REF][START_REF] Sung | Methicillin-Resistant Staphylococcus aureus Endocarditis in an extremely low-birth-weight infant treated with linezolid[END_REF] , three patients suffered from peritonitis [START_REF] Groll | Linezolid treatment of glycopeptideresistant Enterococcus faecium in very low birth weight premature neonates[END_REF] or necrotizing enterocolitis [START_REF] Klingkowski | Linezolid in two premature babies with necrotizing enterocolitis and infection with vancomycin-resistant enterococcus[END_REF] , one patient had nosocomial pneumonia (?) [START_REF] Groll | Linezolid treatment of glycopeptideresistant Enterococcus faecium in very low birth weight premature neonates[END_REF] and one child suffered from a persistent septicaemia [START_REF] Pak | Persistent Staphylococcus capitis septicemia in a preterm infant[END_REF] A c c e p t e d M a n u s c r i p t Dosage and duration of linezolid was according to the manufacturer´s recommendations in 12 of 14 patients. Except for temporary thrombocytopenia in one patient no haematological or other laboratory or clinical side effects of linezolid were observed. In one patient dosage had been increased to 12 mg/kg every 8 without evidence of bone marrow suppression [START_REF] Kumar | Treatment of vancomycin-resistant Enterococcus faecium ventriculitis in a neonate[END_REF] , and even treatment over 9 weeks in one patient was well tolerated [START_REF] Ang | Vancomycin resistant Enterococcus faecium endocarditis in a premature infant successfully treated with linezolid[END_REF] .

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

Conclusion

In clinical studies comparing the efficacy and safety of linezolid versus vancomycin linezolid proved to be well tolerated and as effective as vancomycin in a total of 51 preterm infants. Adding the information out of eight case studies including 14 preterm infants linezolid demontrated to be a save A c c e p t e d M a n u s c r i p t 14

Recommendations

Considering the available data on linezolid in preterm infants we recommend its use as second line antibiotic in preterm infants infected with glycopeptide-resistant bacteria and/or in case of glycopeptide treatment failures. However, in cases of known or suspected glycopeptide resistant pathogens we advise to consider linezolid at an earlier stage of treatment. A dosage regimen of 10 mg/kg body weight given either orally or intravenously every 8 hours in infants aged one week or more and the same dose given every 12 hours in infants younger than one week was shown to be safe and effective with mean treatment duration of 10 to 28 days. Due to the risk of myelosuppression weekly evaluation of complete blood counts is recommended.

Funding

None.

Competing interest

No competing interests are identified by any of the authors.

Ethical approval

This review needed no Ethical Approval.

  , has become an alternative treatment option. Especially in glycopeptide-resistant Gram-positive bacterial infections and in cases of glycopeptide treatment failures linezolid has shown to be a potent second-line drug. Linezolid possesses excellent microbial activity against a wide variety of Gram-positive pathogens, with bactericidal effects against most strains of Streptococcus spp. and bacteriostatic action on Enterococcus spp. and Staphylococcus spp., including vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin Resistant Coagulase-Negative Staphylococcus (MRCNS)

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

 6 We extracted the following types of data from each study identified as specified by our protocol: indications, pharmacokinetics and recommended dosage, duration of treatment, efficacy and side effects.

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

 5 p=0.172) and increases in total bilirubin (linezolid 19.0% vs. vancomycin 10.5%, p=0.405).

  and potent second-line drug in the treatment of nosocomial bacterial infection with multiresistant Gram-positive pathogens in preterm infants. Benefits of linezolid compared to vancomycin include its availability in equivalent intravenous and oral formula and its safer use in combination with aminoglycosides. Linezolid should be used as second-line treatment, but due to overwhelming nosocomial infections in very low birth weight infants its use might be considered earlier in case of unassertive clinical improvement of the baby or knowledge of multiresistant Gram-positive pathogens at the NICU. Study results are limited by the low number of preterm infants included. Further prospective studies are needed to confirm the efficacy and safety of linezolid in the treatment of multiresistant Gram-positive infections in this particular vulnerable group of preterm infants. Additionally information on linezolid levels in plasma and cerebrospinal fluid is warranted.

FiguresFigure 1 :Fig. 1 : 18 .Fig. 1 :

 11181 Figures

  reported the results from a multicenter study of linezolid disposition in preterm and term neonates and young infants. The population of

	42 infants was stratified into 4 subgroups: group 1 (n=9), gestational age <34
	weeks and postnatal age <8 days; group 2 (n=7), gestational age <34 weeks
	and postnatal age 8 days to 12 weeks; group 3 (n=11), gestational age >34
	weeks and postnatal age <8 days; and group 4 (n=15), gestational age >34
	weeks and postnatal age 8 days to 12 weeks. The study was conducted as an

  . Because CL is slower in preterm neonates (gestational age <34 weeks) aged <7 days as well as in term neonates aged <7 days, a longer interval between doses (every 12 hours) is initially recommended in these patients; however, the clinical response should be assessed considering the site and severity of infection and the underlying medical condition. An 8-hour dosing interval may be used in preterm and term neonates under 7 days of life, if the response to linezolid is suboptimal and should be used in all neonates by

7 days of age. This recommendation is supported by the results gained by

Kearns' et al.

in their multicenter study of linezolid disposition in preterm and term neonates and young infants

[START_REF] Kearns | Impact of ontogeny on linezolid disposition in neonates and infants. Pharmacokinetics and Drug Disposition[END_REF]

. Biedenbach and Jones have suggested the following breakpoints for linezolid: minimum inhibitory concentration (MIC)

Table 1 :

 1 Summary of pharmacological studies, studies on tolerability and case studies on linezolid use in preterm children[START_REF] Pharmacia | Zyvox: linezolid injection, linezolid tablets, linezolid for oral suspension package insert[END_REF][START_REF] Kearns | Impact of ontogeny on linezolid disposition in neonates and infants. Pharmacokinetics and Drug Disposition[END_REF][START_REF] Kaplan | Linezolid versus vancomycin for treatment of resistant Gram-positive infections in children[END_REF][START_REF] Deville | Linezolid versus vancomycin for treatment of resistant Grampositive infections in neonates[END_REF][START_REF] Meissner | Hematologic effects of linezolid in young children[END_REF][START_REF] Graham | Linezolid treatment of vancomycin-resistant Enterococcus faecium ventriculitis[END_REF][START_REF] Kumar | Treatment of vancomycin-resistant Enterococcus faecium ventriculitis in a neonate[END_REF][START_REF] Langgartner | Linezolid for treatment of catheterrelated cerebrospinal fluid infections in preterm infants[END_REF][START_REF] Ang | Vancomycin resistant Enterococcus faecium endocarditis in a premature infant successfully treated with linezolid[END_REF][START_REF] Sung | Methicillin-Resistant Staphylococcus aureus Endocarditis in an extremely low-birth-weight infant treated with linezolid[END_REF][START_REF] Groll | Linezolid treatment of glycopeptideresistant Enterococcus faecium in very low birth weight premature neonates[END_REF][START_REF] Klingkowski | Linezolid in two premature babies with necrotizing enterocolitis and infection with vancomycin-resistant enterococcus[END_REF][START_REF] Pak | Persistent Staphylococcus capitis septicemia in a preterm infant[END_REF]

	Year	Age of patients	Number	Dosage	interval	Duration	Adverse	Reference
			Total/preterm	(mg/kg)			events (%)
	2002 PT, T, ≤ 17 y	136/9	10	1 dose	1 d	n. sp.
	2003 PT, T, ≤12 w	42/16	10	1 dose	1 d	n. sp.
	2003 PT, T, ≤ 12 y	215/26	10	8 h	10-28 d	18.8
	2003 PT, T, ≤11 y	215/26	10	8 h	10-28 d	13.1
	2003 PT, T, ≤ 90 d	43/26	10	8 h	10-28 d	7.0
	2002 29 w	1/1	10	8 h	21 d	0
	2007 35 w	1/1	10	8 h	21 d	0
				12	8 h	14 d
	2008 mean 26.4 w	5/5	10	8 h	mean 20.8 d	0
	2003 26 w	1/1	15	8 h	9 w	0
	2008 28 w	1/1	10	8 h	10 d	0
	2006 24 and 30 w	2/2	10	8 h	16/14 d	0
	2004 26 and 28 w	2/2	10	12 h	20 d	50
	PT preterm, T term infant, w weeks, d days, y years,
	n.sp. not specified

Table 2 :

 2 Treatment-related adverse events reported in >1% of 316 hospitalized children including 34 preterm infants <34 weeks of gestational age (randomized 2:1 to linezolid vs. vancomycin) infected with nosocomial multiresistant Gram-positive pathogens[START_REF] Kaplan | Linezolid versus vancomycin for treatment of resistant Gram-positive infections in children[END_REF]

	Adverse Event	Linezolid (n=213)	Vancomycin (n=99)	p-value
	Diarrhea	8 (3.8)*	6 (6.1)	0.3601
	Vomiting	4 (1.9)	1 (1.0)	0.5699
	Thrombocytopenia	4 (1.9)	0 (0)	0.1700
	Loose stools	4 (1.9)	0 (0)	0.1700
	Rash	3 (1.4)	7 (7.1)	0.0082
	Nausea	3 (1.4)	0 (0)	0.2354
	Anemia	3 (1.4)	1 (1.0)	0.7710
	Eosinophilia	3 (1.4)	0 (0)	0.2354
	Oral moniliasis	2 (0.9)	4 (4.0)	0.0634
	Fever	1 (0.5)	3 (3.0)	0.0613
	Red man syndrome	0 (0)	10 (10.1)	<0.0001
	Pruritus	0 (0)	2 (2.0)	0.0374

*numbers in parentheses, percent