Swarna Jaiswal

Brendan Duffy
email: brendan.duffy@dit.ie

Amit Kumar

Niall Stobie

Patrick Mchale

Enhancement of the antibacterial properties of silver nanoparticles using -cyclodextrin as a capping agent

Keywords:

A c c e p t e d M a n u s c r i p t 3 ABSTRACT Silver nanoparticles (AgNPs) were synthesised by reducing silver salts using NaBH 4 followed by capping with varying concentrations of -cyclodextrin (-CD) and were physically characterised. Antibacterial activity against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus was determined by a microtitre well method.

The AgNPs were spherical under transmission electron microscopy, whilst dynamic light scattering showed average diameters of capped particles to be smaller (4-7 nm) than their uncapped equivalents (17 nm). Capped particles demonstrated superior photostability when exposed to intense ultraviolet radiation for 4 h as well as significantly (P < 0.05) higher (up to 3.5-fold) antibacterial activity. The influence of -CD concentration was seen to delay bacterial growth, indicating that a Trojan horse mechanism may be occurring owing to bacterial carbohydrate affinity, thereby enhancing silver ion absorption.

Introduction

The persistence of antibiotic-resistant bacteria has renewed interest in the use of silver and silver-based compounds, including silver nanoparticles (AgNPs), as alternative antibacterial agents. Such compounds can reduce infections in burns patients and prevent bacterial colonisation of prostheses, catheters, dental materials and human skin. The high thermal stability, low toxicity to human cells and effective broad-spectrum antibacterial activity of AgNPs [START_REF] Sharma | Silver nanoparticles: green synthesis and their antimicrobial activities[END_REF] has been exploited in a range of commercial products.

There are several hypotheses to explain the antibacterial activity of AgNPs. Their rapid breakdown releases ionic silver that inactivates vital bacterial enzymes by interacting with essential thiol groups. Silver ions can inhibit bacterial DNA replication, damaging bacterial cytoplasmic membranes, depleting levels of intracellular adenosine triphosphate (ATP) and causing cell death [START_REF] Feng | A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus[END_REF]. The high specific surface-to-volume ratio of AgNPs increases their contact with microorganisms, promoting the dissolution of silver ions, thereby improving biocidal effectiveness. The ability of AgNPs to release silver ions is key to their antibacterial activity [START_REF] Stobie | Prevention of Staphylococcus epidermidis biofilm formation using a lowtemperature processed silver-doped phenyltriethoxysilane sol-gel coating[END_REF].

AgNPs can be synthesised in a number of ways, with borohydride reduction of silver salts being the most common. Stabilisation is achieved using capping agents that bind to the nanoparticle surface and improve stability and water solubility, which are essential to prevent aggregation; examples include water-soluble polymers, A c c e p t e d M a n u s c r i p t 5 oligosaccharides and polysaccharides, sodium dodecyl sulphate (SDS) and sophorolipid (glycolipid) [START_REF] Sing | A direct method for the preparation of glycolipid-metal nanoparticle conjugates: sophorolipids as reducing and capping agents for the synthesis of water redispersible silver nanoparticles and their antibacterial activity[END_REF].

Cyclodextrins (CDs) are a family of soluble, non-toxic molecules consisting of 6, 7 or 8 D-glucopyranosyl residues (denoted as -, -and -CDs, respectively) linked in cyclic structure by -1,4 glycosidic bonds. They can form inclusion complexes incorporating various molecular guests within their hollow, truncated cone-shaped cavity structure, enabling them to be used as drug carriers and enzyme mimics.

Host-guest interaction has been attributed to a combination of weak interactions such as van der Waals forces, hydrogen bonding and hydrophobic interactions [START_REF] Rekharsky | Complexation thermodynamics of cyclodextrins[END_REF]. Of the three cyclodextrins, -CD, with an internal cavity diameter of 0.78 nm, is the most widely used but is too small to entrap AgNPs and simply binds via chemisorption to the nanoparticles through rim hydroxyl groups.

Unmodified -CD has been used to prepare silver and gold nanoparticles in the presence of different reducing agents such as ethanol, dimethylformamide, ethylene glycol and sodium citrate. Little has been published on the antimicrobial activity of unmodified CDs, but modified -CD (dimethylated -CD) inhibited growth of Rhodococcus erythropolis and hydroxypropyl--CD was toxic for Mycoplasma capricolum and Acholeplasma laidlawii, whilst methylated -CD altered growth and cell envelope features of Mycobacterium sp. [START_REF] Donova | Methyl-cyclodextrin alters growth, activity and cell envelope features of steroltransforming mycobacteria[END_REF].

Materials and methods

Synthesis of silver nanoparticles

Aqueous -CD solutions (5, 10 and 20 mM) were stirred into equal volumes of 2 mM AgNO 3 for 15 min and equal volumes of ice-cold 20 mM NaBH 4 were added.

Resultant solutions were stabilised by stirring (24 h). The final molar ratios of -CD:AgNO 3 were 2.5, 5 and 10 to 1, respectively. A -CD-free control was similarly prepared. All chemicals were purchased from Sigma-Aldrich (Dublin, Ireland) and were used without further purification.

Sample characterisation

UV-Vis absorption spectra of -CD-capped AgNPs were acquired in the 300-800 nm wavelength range using an UV-Vis-NIR spectrophotometer (Perkin-Elmer Lambda 900; Perkin-Elmer) operating at a resolution of 2 nm. FT-IR spectra were measured in the 4000-400 cm -1 range with a resolution of 1 cm -1 using a Perkin-Elmer Spectrum GX instrument in transmission mode using potassium bromide (KBr) pellets prepared by mixing KBr powder with AgNP solutions and then drying (24 h, 100 C). AgNP morphology was determined using a JEM-100CX II transmission (340 nm) for 1, 2, 3 and 4 h, respectively. colony-forming units (CFU)/mL] using a Densimat photometer (bioMérieux, France) and diluted with MHB to give a final working concentration of 1  10 6 CFU/mL.

Bacterial strains and growth conditions

Antibacterial activity assay

Antibacterial activities of capped and uncapped AgNPs of different silver concentrations were determined by a microtitre well method [START_REF] Casey | Development of a robust microtiter plate-based assay method for assessment of bioactivity[END_REF]. Samples were doubly diluted in water (100 L) in microtitre wells and bacteria (100 L; 10 6 CFU/mL)

Data analysis 2.5.1. Percentage inhibition of growth

AgNP antibacterial activities were determined by calculating the percentage inhibition of growth using the formula [START_REF] Casey | Development of a robust microtiter plate-based assay method for assessment of bioactivity[END_REF]:

I % =  100
where I is the percentage inhibition of growth, C 18 is the blank-compensated optical density at 600 nm (OD 600 ) of the positive control of the organism at 18 h, C 0 is the blank-compensated OD 600 of the positive control of the organism at 0 h, T 18 is the negative control-compensated OD 600 of the organism in the presence of test sample at 18 h, and T 0 is the negative control-compensated OD 600 of the organism in the presence of test sample at 0 h.

A c c e p t e d

M a n u s c r i p t 9

Kinetic measurement of bacterial growth

Bacterial concentrations in the presence of capped and uncapped AgNPs were determined over time by measuring the OD 600 every 30 min following 30 s of agitation. Antibacterial kinetics was analysed graphically as a plot of contact time versus OD 600 .

Statistical analysis

Statistical differences between multiple comparisons were evaluated using analysis of variance (ANOVA) followed by least significant difference testing. All statistical analyses were carried out using STATGRAPHICS centurion XV (??). A P-value of <0.05 was considered statistically significant.

Results and discussion

Characterisation of -cyclodextrin-capped silver nanoparticles

All AgNPs showed a surface plasmon absorption band around 400 nm as expected for colloidal AgNPs, indicating roughly spherical nanoparticles, whilst solution absorbance intensities increased with -CD concentration. Since the intensity of the plasmon resonance band depends on particle size, shape, metallic material and its surrounding environment, the number of particles cannot be related linearly to the absorbance intensities [START_REF] Slistan-Grijalv | Classical theoretical characterization of the surface plasmon absorption band for silver spherical nanoparticles suspended in water and ethylene glycol[END_REF].

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

Ultraviolet stability

On exposure to intense UV radiation the colour of uncapped AgNPs became darker, with the absorbance peak becoming 14.05 nm broader (with respect to peak width at half maximum) over time. There were no significant changes in absorbance at the higher -CD concentrations, although at 5 mM it increased slightly. This confirmed that uncapped AgNPs continued to grow and/or aggregate over time, whilst AgNPs capped at appropriate -CD concentrations exhibited good stability.

Silver nanoparticle--cyclodextrin binding

FT-IR confirmed -CD binding to the AgNP surface. The free -CD spectrum showed bands at 947 cm -1 (skeletal vibration involving  1-4 linkage) and 756 cm -1 (ring breathing vibration), whilst bands at 708 cm -1 and 579 cm -1 were assigned to pyranose ring vibration [START_REF] Egyed | Spectroscopic studies on -cyclodextrin[END_REF]. Comparison of the spectra of 20 mM -CD-capped

AgNPs and free -CD showed a decrease in the relative intensities of transmittance bands at 947 cm -1 , 756 cm -1 , 708 cm -1 and 579 cm -1 and a suppression of the -OH vibrational band at 3400 cm -1 , indicating deprotonation of the hydroxyl groups [START_REF] Cortés | The chlorhexidine: -cyclodextrin inclusion compound: preparation, characterization and microbiological evaluation[END_REF] at the rim of the CD structure. This suggests that there is chemisorption between the CD molecules and AgNPs via the rim hydroxyl groups. Sorption of CDs might be due to binding of the AgNPs to hydroxyl groups at positions C 2 or C 3 . The oxyanion thus formed is less nucleophilic than the non-deprotonated hydroxyl at position C 6 [START_REF] Cruz | Synthesis of magnetite nanoparticles--cyclodextrin complex[END_REF] and is therefore more likely to bind to the AgNPs. and 20 mM, respectively) and 16.3 nm for the uncapped particle. The capped AgNPs were much larger in size than the -CD cavity (0.78 nm), indicating chemisorption interaction through the CD rim hydroxyls rather than encapsulation.

Antibacterial activity

Most published AgNP antibacterial studies have used plate counting [START_REF] Pal | Does the antibacterial activity of silver nanoparticles depend on the shape of the nanoparticle? A study of the Gram-negative bacterium Escherichia coli[END_REF] and agar diffusion methods. Fewer reports have used the quantitative microtitre well dilution method. The effect of -CD capping on antibacterial activity was determined by comparing the percentage inhibition of bacterial growth by uncapped AgNPs compared with that of capped AgNPs (5, 10 and 20 mM -CD) using a range of Ag concentrations (100, 50, 25, 12.5, 6.25 and 3.12 ppm) detailed in Table 1.

-CD-capped AgNPs displayed significantly (P < 0.05) greater antibacterial activity against P. aeruginosa than uncapped AgNPs. AgNPs capped with 20 mM and 10 mM -CD had the strongest activity of 99.2% and 97.3% inhibition, respectively, at Antibacterial activity increased with corresponding rises in both silver and cyclodextrin concentrations in all cases. Importantly, -CD alone showed no antibacterial activity.

Bacterial growth curves showed that the AgNPs delayed growth. The 20 mM and 10 mM -CD-capped AgNPs containing a [Ag] of 6.25 ppm completely inhibited P.

aeruginosa growth until 18 h (Fig. 1a) compared with ca. 10 h for the uncapped AgNPs. Even at a [Ag] of 3.12 ppm the 20 mM -CD-capped AgNPs were more effective than uncapped AgNPs (Fig. 1b). The 20 mM -CD-capped AgNPs at a [Ag] of 6.25 ppm exhibited superior antibacterial activity against E. coli compared with uncapped AgNPs, delaying growth for 9 h versus 4.5 h (Fig. 1c). A similar trend was observed for the 10 mM and 5 mM -CD-capped AgNPs. At a [Ag] of 6.25 ppm, AgNPs capped with 20 mM and 10 mM -CD delayed growth of S. aureus for 17 h (Fig. 1e). At a [Ag] of 3.12 ppm capping doubled growth inhibition (Fig. 1f). Overall, The increased antibacterial activity of -CD-capped AgNPs may be attributed to a biodegradable capping agent forming smaller nanoparticles without aggregation.

These have a high surface-to-volume ratio and undergo a higher level of interaction with the bacterial cell surface than the larger uncapped AgNPs, resulting in a higher antibacterial activity.

It is proposed that intimate contact between AgNPs and organisms may enhance the transfer of Ag ions to the bacterial cell, whilst bacterial degradation of the -CD saccharide promotes the release of silver ions (Fig. 2) [START_REF] Loher | Micro-organismtriggered release of silver nanoparticles from biodegradable oxide carriers allows preparation of self-sterilizing polymer surfaces[END_REF]. Such interactions have been described as a Trojan-horse mechanisms and have been reported in the literature where fetal bovine serum was used to improve AgNP efficacy [START_REF] Park | Silver nanoparticles induce cytotoxicity by a Trojan-horse type mechanism[END_REF]. These results are in accordance with a previous report in which AgNPs synthesised by disaccharides had higher antibacterial activities than those synthesised by monosaccharides [START_REF] Panáček | Silver colloid nanoparticles: synthesis, characterization, and their antibacterial activity[END_REF].

Conclusion

In summary, there was an increase in the antibacterial activity of -CD-capped

AgNPs compared with their uncapped equivalents. This activity increased as the concentration of -CD increased. Thus, -CD capping can be used to achieve higher antibacterial efficacies at lower metal ion concentrations.

  The present work investigated AgNP synthesis using different concentrations of unmodified -CD with NaBH 4 as the reducing agent. AgNPs were characterised by

  (UV-Vis) spectrometry, Fourier transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS) and transmission electron microscopy (TEM). Antibacterial activities of uncapped and -CD-capped AgNPs against Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria were determined.

A

  c c e p t e d M a n u s c r i p t 7 electron microscope (JEOL) operated at an accelerating voltage of 100 kV. Samples were drop-cast onto standard Formvar-coated copper grids (200-300 nm) and then air dried. The average particle size (hydrodynamic diameter) was determined at 25 C by DLS using a Malvern Zetasizer Nano instrument (Malvern Instruments) equipped with a 4 mW He-Ne laser and measured using an automatic mode with a scattering angle of 90. Sample photostability was determined by exposing solutions to an intense UV source (Q-sun Xenon Test Chamber; Q-Lab) operating at 0.4 W/m 2

  Gram-negative bacteria (E. coli ATCC 11229 and P. aeruginosa ATCC 27852) and Gram-positive bacteria (S. aureus ATCC 25923) were grown, subcultured and maintained on Mueller-Hinton agar (Lab M) and stored at 4 C. For the experiment, a single colony of each organism was inoculated into 10 mL of Mueller-Hinton broth (MHB) and incubated overnight at 37 C with shaking at 200 rpm. The optical density of the overnight culture was adjusted to that of a 0.5 McFarland standard [1.5  10 8

  A c c e p t e d M a n u s c r i p t 8 were added. Negative (AgNPs 100 L + sterile MHB 100 L) and positive (sterile MHB 100 L + bacterial suspension 100 L) control wells and a sterility control blank (sterile MHB 200 L) were included in each assay. Plates were incubated (18 h) in a microtitre plate reader (PowerWave TM Microplate spectrophotometer; BioTek, Winooski, VT) at 37 C. Samples were tested in duplicate on each plate and each plate was analysed in triplicate.

A c c e p t e d M a n u s c r i p t 11 3. 4 .

 114 Particle sizing TEM micrographs of capped and uncapped AgNPs showed them to be nearly spherical in shape, with the average size decreasing with increasing -CD capping. DLS showed particles sizes of 6.2, 5.1 and 4.0 nm for -CD-capped AgNPs[START_REF] Rekharsky | Complexation thermodynamics of cyclodextrins[END_REF][START_REF] Cortés | The chlorhexidine: -cyclodextrin inclusion compound: preparation, characterization and microbiological evaluation[END_REF]

12. 5

 5 ppm [Ag], whereas inhibition for AgNPs capped with 5 mM -CD was 64.5% and for uncapped AgNPs was 13.9%. Both capped and uncapped AgNPs with a [Ag] 25 ppm significantly inhibited bacterial growth, whilst those with a [Ag] 6.25 ppm showed no inhibition.

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

 12 coli was 97% inhibited at a [Ag] of 12.5 ppm. Below this there was a variation in inhibition, with 20 mM -CD-capped AgNPs achieving 53.5% inhibition at 6.25 ppm, whereas uncapped AgNPs showed only 22.8% inhibition.Staphylococcus aureus was 97.1% inhibited by 20 mM -CD-capped AgNPs at a [Ag] of 6.25 ppm compared with 59.5% inhibition by uncapped AgNPs. Antibacterial activity was high (94.0% inhibition) even at a concentration of 10 mM -CD. Similar findings were obtained with a [Ag] of 3.12 ppm.

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

 13 duration of the lag growth phase of the test organisms when grown in the presence of AgNPs with different [Ag] increased as the -CD capping concentration increased.

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 17 Fig. 1 .Fig. 2 .

 1712 Fig. 1. Growth curves of (a,b) Pseudomonas aeruginosa in the presence of 6.25 ppm

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

Table 1

 1 Percentage growth inhibition of Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus in the presence of uncapped and -cyclodextrin (-CD)-capped(5, 10 and 20 mM) silver nanoparticles (AgNPs) containing different silver P. aeruginosa 20 mM -CD-AgNPs 99.72 ± 0.2 96.11 ± 3.7 98.36 ± 1.1 99.24 ± 0.5 N/D N/D P. aeruginosa 10 mM -CD-AgNPs 99.08 ± 0.8 98.59 ± 1.5 98.34 ± 1.4 97.27 ± 0.7 N/D N/D P. aeruginosa 5 mM -CD-AgNPs 99.85 ± 0.4 97.23 ± 1.5 98.37 ± 1.1 64.50 ± 6.2 N/D N/D AgNPs 99.76 ± 0.3 97.75 ± 2.6 96.99 ± 1.2 97.48 ± 0.8 97.14 ± 0.9 46.96 ± 14.6 S. aureus 10 mM -CD-AgNPs 99.06 ± 0.8 97.50 ± 0.8 98.14 ± 1.6 97.4 ± 0.8 93.97 ± 3.2 26.68 ± 10.5

	Edited Table 1
	concentrations a
	Microorganism Sample	[Ag]
			100 ppm	50 ppm	25 ppm	12.5 ppm	6.25 ppm	3.12 ppm
	P. aeruginosa Uncapped AgNPs	97.40 ± 2.2 96.55 ± 1.3 96.23 ± 3.1 13.92 ± 6.6 N/D	N/D
	E. coli	20 mM -CD-AgNPs 99.19 ± 0.3 96.81 ± 2.9 96.91 ± 1.6 98.40 ± 0.7 53.45 ± 4.8 34.84 ± 5.8
	E. coli	10 mM -CD-AgNPs 100 ± 0	96.96 ± 2.9 97.01 ± 1.4 97.02 ± 0.6 43.66 ± 3.3 20.09 ± 4.6
	E. coli	5 mM -CD-AgNPs 99.59 ± 0.4 97.38 ± 3.3 97.91 ± 2.0 97.39 ± 0.2 29.28 ± 10.9 19.38 ± 3.2
	E. coli	Uncapped AgNPs	100 ± 0	96.52 ± 2.4 97.73 ± 0.7 97.58 ± 2.3 22.75 ± 10.3 17.3 ± 1.0
	S. aureus 20 mM -CD-S. aureus 5 mM -CD-AgNPs 98.62 ± 1.0 98.46 ± 1.5 97.04 ± 0.9 96.98 ± 0.9 66.58 ± 12.8 18.245 ± 6.3
	S. aureus	Uncapped AgNPs	98.40 ± 0.8 97.31 ± 1.9 97.46 ± 1.0 96.88 ± 0.9 59.50 ± 7.6 16.97 ± 7.6


Axel Dalhoff
email: adalhoff@t-online.de

Sabine Schubert

Dichotomous selection of high-level oxacillin resistance in Staphylococcus aureus by fluoroquinolones

Keywords:

second, by exposing the heteroresistant MRSA P8 parent strain as well as fluoroquinolone-resistant subpopulations derived from strain P8 to constant fluoroquinolone concentrations ranging from 0.015 mg/L to 128 mg/L; and third, by exposing the heteroresistant MRSA population of strain P8 to fluctuating concentrations of ciprofloxacin, levofloxacin and moxifloxacin simulating oral doses of 500 mg twice a day, 500 mg once daily (qd) and 400 mg qd, respectively, compared with amoxicillin/clavulanic acid 500 mg three times a day. Total viable counts and subpopulations resistant to 2, 4 and 8 the fluoroquinolone MICs and to 32, 64 and 128 mg/L oxacillin [high-level oxacillin (hl-OXA)-resistant] were quantitated. None of the fluoroquinolones acted as a mutator; ciprofloxacin and levofloxacin selected for hl-OXA resistance, whereas moxifloxacin selected towards hl-OXA resistance by one order of magnitude less frequently. The P8 parent and fluoroquinoloneresistant subpopulations were eliminated by ciprofloxacin or levofloxacin A c c e p t e d M a n u s c r i p t

Introduction

Meticillin resistance leading to treatment failure was detected 1 year after its launch [START_REF] Jevons | Celbenin-resistant staphylococci[END_REF][START_REF] Knox | Celbenin-resistant staphylococci[END_REF]. Shortly after the identification of meticillin resistance, it became apparent that 'resistant clones' were not true mutants but represented a minority population of a genetically uniform culture [START_REF] Lacey | The mechanism of methicillin resistance[END_REF]. It is characteristic for most meticillin-resistant Staphylococcus aureus (MRSA) that expression of meticillin resistance is heterogeneous; most cells express a low level of resistance, from which a small subpopulation of highly resistant subclones segregate at a frequency of 10 -7 to 10 -3 [START_REF] Berger-Bächi | Factors influencing methicillin resistance in staphylococci[END_REF]. Therefore, phenotypic expression of meticillin resistance is highly variable and each strain has a characteristic profile of meticillin resistance heterogeneity [START_REF] Tomasz | Stable classes of phenotypic expression in methicillin-resistant clinical isolates of staphylococci[END_REF]. The mechanism of meticillin resistance is due to the acquisition of an altered penicillin-binding protein 2A (PBP2A) [START_REF] Jevons | Celbenin-resistant staphylococci[END_REF][START_REF] Utsui | Role of an altered penicillin-binding protein in methicillin-and cephem-resistant Staphylococcus aureus[END_REF] encoded by the mecA gene, which is located on the so-called staphylococcal cassette chromosome mec (SCCmec) also containing additional genes for antibiotic resistance elements encoding for aminoglycoside, tetracycline and macrolide-lincosamide-streptogramin B (MLS B ) resistance, insertion sequences as well as genes of uncertain function [START_REF] Berger-Bächi | Factors influencing methicillin resistance in staphylococci[END_REF][START_REF] Deresinski | Methicillin-resistant Staphylococcus aureus: an evolutionary, epidemiologic, and therapeutic odyssey[END_REF]. Therefore, these drug classes can act as co-selectors for MRSA.

A steady rise in the proportion of S. aureus resistant to meticillin occurred in the 1960-1970s, but inexplicably the numbers fell between the latter half of the 1970s and the early 1980s. The reasons for this decline are not clear. On the one hand there was a continuous and moderately heavy use of antibiotics such as isoxazolyl penicillins (flucloxacillin, nafcillin, dicloxacillin, etc.), which have been implicated as a selective pressure for MRSA. On the other hand, A c c e p t e d M a n u s c r i p t aminoglycosides to which many MRSA were susceptible at that time were increasingly prescribed. In the 1980s, a rise in gentamicin-resistant MRSA was reported. Not only do aminoglycosides act as co-selectors for MRSA [START_REF] Johnson | Surveillance and epidemiology of MRSA bacteraemia in the UK[END_REF][START_REF] Marples | Methicillin-resistant Staphylococcus aureus in England and Wales[END_REF][START_REF] Shanson | Antibiotic-resistant Staphylococcus aureus[END_REF][START_REF] Hawkey | The growing burden of antimicrobial resistance[END_REF][START_REF] Reynolds | Superbugs and superdrugs: a history of MRSA[END_REF] but use of cephalosporins, penicillins, carbapenems, macrolides, vancomycin, metronidazole and fluoroquinolones has been important in selecting for MRSA [START_REF] Johnson | Dominance of EMRSA-15 and -16 among MRSA causing nosocomial bacteraemia in the UK: analysis of isolates from the European Antimicrobial Resistance Surveillance System (EARSS)[END_REF][START_REF] Dziekan | Methicillin-resistant Staphylococcus aureus in a teaching hospital: investigation of nosocomial transmission using a matched case-control study[END_REF][START_REF] Weber | Fluoroquinolones and the risk for methicillin-resistant Staphylococcus aureus in hospitalised patients[END_REF][START_REF] Graffunder | Risk factors associated with nosocomial methicillin-resistant Staphylococcus aureus (MRSA) infection including previous use of antimicrobials[END_REF]. Although a causal relationship between antimicrobial drug use and MRSA colonisation and/or infection has been demonstrated [START_REF] Salgado | Community-acquired methicillinresistant Staphylococcus aureus: a meta-analysis of prevalence and risk factors[END_REF][START_REF] Harbarth | Universal screening for methicillin-resistant Staphylococcus aureus at hospital admission and nosocomial infection in surgical patients[END_REF][START_REF] Tacconelli | Does antibiotic exposure increase the risk of methicillin-resistant Staphylococcus aureus (MRSA) isolation? A systematic review and meta-analysis[END_REF], the evidence is not conclusive.

In any consideration of the selective effect of antibiotic regimens, not only the resistance mechanisms of each antibiotic/organism combination should be considered. It is also of clinical relevance if any agent of a given drug class may exert the same or differential selective pressure. Furthermore, the problem of co-selection of strains has to be considered, as some of the SCCmec islands contain genes encoding for aminoglycoside, tetracycline and MLS B resistance [START_REF] Deresinski | Methicillin-resistant Staphylococcus aureus: an evolutionary, epidemiologic, and therapeutic odyssey[END_REF]. In addition, selective pressures in the hospital environment are very complex as different intensities of care are used for different patients [START_REF] Dziekan | Methicillin-resistant Staphylococcus aureus in a teaching hospital: investigation of nosocomial transmission using a matched case-control study[END_REF][START_REF] Weber | Fluoroquinolones and the risk for methicillin-resistant Staphylococcus aureus in hospitalised patients[END_REF][START_REF] Graffunder | Risk factors associated with nosocomial methicillin-resistant Staphylococcus aureus (MRSA) infection including previous use of antimicrobials[END_REF] and different policies for hospital cleaning and hygienic measures are applied [START_REF] Dancer | Importance of the environment in methicillin-resistant Staphylococcus aureus acquisition: the case for hospital cleaning[END_REF][START_REF] Dancer | The role of environmental cleaning in the control of hospital-acquired infection[END_REF].

A further complication is the recent recognition that some community-acquired (CA)-MRSA may originate in hospitals and that novel strains of MRSA can evolve by horizontal gene transfer of the mecA gene and then be subjected to selective pressure.

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

Although antibacterials in general select for MRSA, fluoroquinolones in particular are considered to act as mutators or selectors for meticillin resistance in S. aureus [START_REF] Crowcroft | Methicillin-resistant Staphylococcus aureus and antimicrobial use in Belgian hospitals[END_REF][START_REF]MRSA-Bedrohung durch Antibiotikatherapie?[END_REF]. Therefore, three questions to be addressed are: is fluoroquinolone use per se linked to the emergence of MRSA? Do fluoroquinolones act as selectors of or mutators for meticillin resistance in S. aureus? And if so, may different fluoroquinolones be characterised by a differential selective potential?

Materials and methods

Strains, media and agents studied

Twenty-four strains each of healthcare-associated (HA) meticillin-susceptible S. aureus (MSSA) and MRSA as well as six CA-MRSA were studied. All strains were shown to be negative or positive for mecA by hybridisation. All strains had recently been isolated and characterised at the University Hospitals in Muenster (Germany) and Zürich (Switzerland). Fluoroquinoloneresistant MRSA were generated in the laboratory as described previously [START_REF] Dalhoff | Effect of pH on the in vitro activity of and propensity for emergence of resistance to fluoroquinolones, macrolides, and a ketolide[END_REF].

Briefly, six HA-MRSA were grown overnight in brain-heart infusion (BHI) broth guidelines [START_REF]Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically[END_REF]. BHI agar was inoculated with 1  10 5 colony-forming units (CFU)/mL and was incubated for 20 h at 36  1 C in ambient air.

Ciprofloxacin and moxifloxacin were provided by Bayer Healthcare (Leverkusen, Germany). Levofloxacin (Sanofi-aventis Deutschland GmbH, Frankfurt, Germany) and amoxicillin/clavulanic acid (fixed 4:1 combination; GlaxoSmithKline GmbH & Co. KG, Munich, Germany) were obtained from commercially available sources. All agents were of defined activity.

Spontaneous emergence of meticillin or fluoroquinolone resistance

Population analysis profiling

One heteroresistant MRSA strain (S. aureus P8) was used for these experiments. From this strain, fluoroquinolone-resistant subpopulations resistant to 2, 4 and 8 the MIC of either ciprofloxacin (initial MIC = 0.25 mg/L), levofloxacin (initial MIC = 0.25 mg/L) or moxifloxacin (initial MIC = 0.125 mg/L) were elicited by incubating ca. 10 log 10 CFU/mL in tubes containing rising concentrations of either fluoroquinolone. From the culture containing the highest drug concentration permitting visible growth following an incubation period of 48 h, an aliquot was transferred as a 1:20 dilution to inoculate a second set of serial fluoroquinolone dilutions. After 48 h incubation, the dilution procedure was repeated again until subpopulations were elicited that were resistant to the multiples of the initial individual fluoroquinolone MICs.

The parent strain and the three either ciprofloxacin-, levofloxacin-or moxifloxacin-resistant subpopulations were grown to ca. 9 log 10 CFU/mL in BHI broth for 20 h at 37 C. The final inoculum of ca. 11 log 10 CFU/mL was obtained by centrifugation and re-suspension of the test strain in fresh medium. Aliquots were then plated onto BHI agar plates containing a fluoroquinolone concentration range (one fluoroquinolone concentration per plate) of 0.015, 0.025, 0.03, 0.05, 0.06, 0.075, 0.10, 0.12, 0.15, 0.20, 0.24, Plates were incubated in ambient air at 36  1 C for 20 h. Thus, MRSA strain P8 was exposed to 37 fluoroquinolone concentrations. Total viable counts were quantitated on drug-free agar plates as well as on plates containing 2% NaCl and 128 mg/L oxacillin. Plates were controlled after 24 h, but colonies were counted after an incubation period of 48 h at 37 C (as colonies were not clearly visible after 24 h). Population analysis profiling was used to determine whether the three different fluoroquinolones studied exhibited a differential bactericidal activity against three different fluoroquinolone-resistant subpopulations of a heteroresistant MRSA indicator strain.

In vitro pharmacokinetic simulation model

One heteroresistant MRSA strain (P8) was used for these experiments. A slightly modified one-compartment model according to and illustrated by Grasso et al. [START_REF] Grasso | New in vitro model to study the effect of antibiotic concentration and rate of elimination on antibacterial activity[END_REF] was used. Briefly, this model consists of a central compartment (calibrated flasks only were used for all experiments) that is placed on heated magnetic stirrer. The antibacterial is pumped via a programmable pump into the central compartment until the maximum serum concentration (C max ) to be simulated is reached at T max . Thereafter, drug-free medium is pumped by a second programmable pump into the central compartment and is continuously eliminated in parallel to mimic elimination half-life (t 1/2 ) values. Control growth in the absence of antibiotics is studied in the same model under identical conditions. To avoid wash-out of bacteria from the central compartment when simulating fluctuating concentration Actual drug concentrations achieved in this model were on average within 4.2% of the desired profile, which is in agreement with previous studies [START_REF] Schubert | Pharmacodynamics of moxifloxacin and levofloxacin simulating human serum and lung concentrations[END_REF].

Interday and intraday reproducibility ranged from 92.4% to 98.5% as qualified calibrated flasks were used.

The following pharmacokinetic (PK) parameters calculated on the basis of total drug concentrations were used as a basis for the PK simulations: for A c c e p t e d M a n u s c r i p t amoxicillin 500 mg three times a day, C max = 11.0 mg/L, T max = 2.0 h, t 1/2 = 3.5 h, area under the concentration-time curve (AUC) = 55.4 mg h/L and protein binding (PB) = 20% [START_REF] Dalhoff | Studies on the pharmacokinetics of amoxicillin after intravenous, intramuscular and oral administration [in German][END_REF][START_REF] Todd | Amoxicillin/clavulanic acid. An update of its antibacterial activity, pharmacokinetic properties and therapeutic use[END_REF]; for ciprofloxacin 500 mg twice a day, C max = 2.5 mg/L, T max = 1.5 h, t 1/2 = 5 h, AUC = 22.3 mg h/L and PB = 30% [START_REF] Dudley | Pharmacokinetics of fluoroquinolones[END_REF]; for levofloxacin 500 mg once daily (qd), C max = 6.6 mg/L, T max = 1.3 h, t 1/2 = 8.0 h, AUC = 57.3 mg h/L and PB = 30% [START_REF] Dudley | Pharmacokinetics of fluoroquinolones[END_REF]; and for moxifloxacin 400 mg qd, C max = 3.3 mg/L, T max = 1.5 h, t 1/2 = 13 h, AUC = 30 mg h/L and PB = 40% [START_REF] Dudley | Pharmacokinetics of fluoroquinolones[END_REF][START_REF] Stass | Pharmacokinetics and elimination of moxifloxacin after oral and intravenous administration in man[END_REF]. A c c e p t e d M a n u s c r i p t

Results

MRSA strains in the strict sense (mecA-positive but fluoroquinolone susceptible) are as susceptible to the three fluoroquinolones studied as MSSA strains (Table 1). Furthermore, the spontaneous mutation frequencies towards fluoroquinolone resistance were almost the same amongst the CA-MRSA, HA-MRSA and MSSA strains studied as well as the three laboratorygenerated fluoroquinolone-resistant MRSA strains and the fluoroquinoloneresistant strain P8 (Table 1). Strains with 4 the initial moxifloxacin MIC could be elicited in <50% of the isolates studied, whereas ciprofloxacin or levofloxacin resistance emerged in all isolates studied (Table 1).

High-level meticillin resistance emerged amongst the strains studied at a low frequency. Pre-existing highly meticillin-resistant subclones emerged spontaneously in the HA-MRSA but not the CA-MRSA controls at a frequency of 1-5  10 -7 and were selected by amoxicillin/clavulanic acid at a frequency of 1-2  10 -2 to 2.4  10 -4 . Ciprofloxacin and levofloxacin selected for high-level meticillin resistance by one to two orders of magnitude and moxifloxacin by three to four orders of magnitude less frequently than amoxicillin/clavulanic acid (Table 1). 2). For the sake of clarity, emergence of highly meticillin-resistant subclones upon exposure to 1, 5, 10, 50 and 100 mg/L of either ciprofloxacin, levofloxacin or moxifloxacin only are summarised in Table 2.

Selection of highly meticillin

Exposure of the indicator strain S. aureus P8 to a broad range of constant ciprofloxacin and levofloxacin concentrations resulted in a moderately concentration-dependent reduction of the initial inoculum. The inoculum of the parent population of S. aureus P8 was eradicated by 12.8 mg/L ciprofloxacin; the inoculum of the subpopulations resistant to 2 the ciprofloxacin MIC was eradicated by 38.4 mg/L ciprofloxacin; whereas the inoculated viable counts of the subpopulations being resistant to 4 and 8 the MIC were reduced by ca. 9.2 and 6.0 log CFU/mL by 128 mg/L ciprofloxacin.

Likewise, the inoculum of the parent population of S. aureus P8 was eradicated by 3.8 mg/L levofloxacin; the inocula of the subpopulations resistant to 2 and 4 the levofloxacin MICs were eradicated by 12.8 mg/L and 31.0 mg/L levofloxacin, respectively; whereas the inoculated viable counts of the subpopulation resistant to 8 the MIC was reduced by ca. 10.4 log CFU/mL by 128 mg/L levofloxacin. In contrast, moxifloxacin eradicated the inocula of the parent strain as well as the three moxifloxacin-resistant subpopulations at moxifloxacin concentrations of 0.30, 0.60, 1.2 and 2.4 mg/L, respectively.

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

The frequencies with which highly meticillin-resistant subclones emerged decreased in parallel with the reduction of total viable counts by increasing fluoroquinolone concentrations (Table 2). Ciprofloxacin selected more highly meticillin-resistant subclones than levofloxacin; moxifloxacin exerted no selective potential.

The MRSA selective potential of fluoroquinolones was analysed in an in vitro PK simulation model. Again, the MRSA heteroresistant strain P8 served as the indicator strain. Fluctuating amoxicillin/clavulanic acid concentrations, serving as a control, selected rapidly for resistance (Fig. 2). Ciprofloxacin rapidly selected both the ciprofloxacin-resistant (within 24 h) as well as the three oxacillin-resistant subpopulations. CFUs of the three pre-existing hl-MRSA subpopulations (32, 64 and 128 mg/L oxacillin) remained constant for ca. 36 h. Thereafter, a continuous increase in CFUs of the oxacillin-resistant subpopulations was noted (Fig. 3). Levofloxacin was also a rapid selector of fluoroquinolone resistance. The three oxacillin-resistant subpopulations were selected from 48 h onwards and less rapidly than by ciprofloxacin (Fig. 4).

Moxifloxacin, however, eliminated all the hl-MRSA subpopulations of S. aureus P8, i.e. the fluoroquinolone-resistant as well as the heteroresistant MRSA subpopulations, within 36 h, so that the in vitro PK simulation model was sterilised (Fig. 5).

A c c e p t e d

M a n u s c r i p t

Discussion

Data generated in this study demonstrate that fluoroquinolones do not act as mutators towards hl-OXA resistance. However, ciprofloxacin and levofloxacin are potent selectors, whereas moxifloxacin is a poor selector. Thus, fluoroquinolones exert a dichotomous MRSA-selective potential in heteroresistant MRSA. This finding is in good agreement with data generated by Venezia et al. [START_REF] Venezia | Selection of high-level oxacillin resistance in heteroresistant Staphylococcus aureus by fluoroquinolones exposure[END_REF]. Fluoroquinolone-susceptible heteroresistant MRSA were exposed to constant subinhibitory concentrations of ciprofloxacin, levofloxacin, gatifloxacin and moxifloxacin. All four fluoroquinolones selected for hl-OXA resistance; however, the proportion of hl-OXA-resistant MRSA was one to three orders of magnitude greater following ciprofloxacin exposure than upon exposure to the two 8-methoxy fluoroquinolones [START_REF] Venezia | Selection of high-level oxacillin resistance in heteroresistant Staphylococcus aureus by fluoroquinolones exposure[END_REF]. Furthermore, the MICs of the strains studied in this series of experiments, the mutational frequencies and the bactericidal activities are in good agreement with previously published data [START_REF] Dalhoff | In vitro antibacterial activity and pharmacodynamics of new quinolones[END_REF][START_REF] Thauvin-Eliopoulos | Activity in vitro of the quinolones[END_REF][START_REF] Schmitz | Activity of quinolones against Gram-positive cocci: mechanisms of drug action and bacterial resistance[END_REF][START_REF] Goldstein | Virulence characteristics of community-associated Staphylococcus aureus and in vitro activities of moxifloxacin alone and in combination against community-associated and healthcare-associated meticillin-resistant andsusceptible S. aureus[END_REF][START_REF] Dalhoff | In vitro activity of BAY 12-8039, a new 8-methoxyquinolone[END_REF].

The low MRSA-selective potential of moxifloxacin in contrast to the high selective potential of ciprofloxacin and levofloxacin is likely due to the high and pronounced bactericidal activity of moxifloxacin against MSSA and MRSA, whereas ciprofloxacin and levofloxacin reduce viable counts of S. aureus much more slowly as described in this study and as published previously [START_REF] Salgado | Community-acquired methicillinresistant Staphylococcus aureus: a meta-analysis of prevalence and risk factors[END_REF][START_REF] Dalhoff | In vitro activity of BAY 12-8039, a new 8-methoxyquinolone[END_REF][START_REF] Schaper | Kinetics and quantification of antibacterial effects of -lactams, macrolides and quinolones against Gram-positive and Gram-negative RTI pathogens[END_REF][START_REF] Kayser | Comparative activity of moxifloxacin and other quinolones against staphylococci [in German[END_REF].

Clinical use of early quinolones like levofloxacin as opposed to the use of recent quinolones like moxifloxacin in the treatment of Gram-positive A c c e p t e d M a n u s c r i p t infections in indications such as community-acquired respiratory tract infections or skin and skin-structure infections may trigger the selection of quinolone-resistant MRSA (see above and [START_REF] Weber | Fluoroquinolones and the risk for methicillin-resistant Staphylococcus aureus in hospitalised patients[END_REF][START_REF] Venezia | Selection of high-level oxacillin resistance in heteroresistant Staphylococcus aureus by fluoroquinolones exposure[END_REF][START_REF] Graffunder | Risk factors associated with nosocomial methicillin-resistant Staphylococcus aureus (MRSA) infection including previous use of antimicrobials[END_REF]). Furthermore, MRSA tended to develop fluoroquinolone resistance more frequently than MSSA (Table 1), which is in agreement with previous data [START_REF] Venezia | Selection of high-level oxacillin resistance in heteroresistant Staphylococcus aureus by fluoroquinolones exposure[END_REF]. This phenomenon may be due to the fact that on the chromosomal map of the S. aureus genome the mecA gene is located between protein A and DNA gyrase genes. Therefore, mutations in the gyrase may have an effect on the expression of mecA in heteroresistant MRSA strains [START_REF] Venezia | Selection of high-level oxacillin resistance in heteroresistant Staphylococcus aureus by fluoroquinolones exposure[END_REF] and some cell wall-associated proteins such a protein A and fibronectin-binding proteins [START_REF] Bisognano | Increased expression of fibronectin-binding proteins by fluoroquinolone-resistant Staphylococcus aureus exposed to subinhibitory levels of ciprofloxacin[END_REF]43].

In conclusion, the experimental data of this study and the clinical findings [START_REF] Weber | Fluoroquinolones and the risk for methicillin-resistant Staphylococcus aureus in hospitalised patients[END_REF][START_REF] Venezia | Selection of high-level oxacillin resistance in heteroresistant Staphylococcus aureus by fluoroquinolones exposure[END_REF][START_REF] Graffunder | Risk factors associated with nosocomial methicillin-resistant Staphylococcus aureus (MRSA) infection including previous use of antimicrobials[END_REF]

A

  c c e p t e d M a n u s c r i p t ABSTRACT The objective of this study was to determine whether exposure of Staphylococcus aureus to early (ciprofloxacin or levofloxacin) and a recent fluoroquinolone (moxifloxacin) has differential potential as a mutator or selector for meticillin resistance. The potential of fluoroquinolones to act as mutators or selectors was studied in 24 strains each of healthcare-associated meticillin-susceptible S. aureus (MSSA) and meticillin-resistant S. aureus (MRSA) as well as 6 strains of community-acquired MRSA. Mutator or selector potential was studied first by exposing isolates to 0.5 the fluoroquinolone minimal inhibitory concentration (MIC) and screening for either single-step fluoroquinolone resistance or high-level oxacillin resistance;

A

  c c e p t e d M a n u s c r i p t concentrations >10-fold higher than the MICs, whereas moxifloxacin eliminated all subpopulations by concentrations 2-3-fold the MIC. Finally, exposure of P8 to fluctuating amoxicillin/clavulanic acid, ciprofloxacin and levofloxacin concentrations, respectively, caused a rapid selection of fluoroquinolone and hl-OXA resistance. Moxifloxacin reduced total viable counts rapidly, thus preventing the emergence of resistant subpopulations. In conclusion, fluoroquinolones do not act as mutators towards hl-OXA resistance. However, ciprofloxacin and levofloxacin are potent selectors of hl-OXA resistance, whereas moxifloxacin is a poor selector. In contrast to ciprofloxacin and levofloxacin, moxifloxacin exerts a high bactericidal activity against staphylococci, thus minimising the probability for selection of resistance. Thus, fluoroquinolones exert a dichotomous MRSA-selective potential in heteroresistant MRSA.

(

  Oxoid, Wesel, Germany) containing doubling dilutions of either ciprofloxacin, levofloxacin or moxifloxacin. From the culture containing the highest drug concentration permitting visible bacterial growth [i.e. 0.5 the minimal inhibitory concentration (MIC)], an aliquot was transferred as a 1:20 dilution to inoculate a second set of serial fluoroquinolone dilutions. Following overnight incubation, the dilution procedure was repeated again until the initial fluoroquinolone MIC increased eight-fold. MICs were determined by the agar A c c e p t e d M a n u s c r i p t dilution method according to Clinical and Laboratory Standards Institute

  Single-step fluoroquinolone resistance testing was performed as outlined by Dalhoff et al.[START_REF] Dalhoff | Effect of pH on the in vitro activity of and propensity for emergence of resistance to fluoroquinolones, macrolides, and a ketolide[END_REF]. To characterise the spontaneous emergence of fluoroquinolone resistance, each of the 24 MSSA and MRSA strains (inoculum 1  10 9 -10 10 CFU/mL) were spread over BHI agar plates incorporating the study drugs (ciprofloxacin, moxifloxacin or levofloxacin ) at 4 MIC of the individual test organisms. Following overnight incubation at 36  1 C, the frequency of spontaneous resistance was determined by colony counting.Emergence of high-level oxacillin (hl-OXA) resistance in the 24 strains each of MSSA and MRSA was tested in the presence of 0.5 MIC of the three fluoroquinolones as well as amoxicillin (positive control) and saline (negative control) by incorporating 128 mg/L oxacillin into agar containing 2% NaCl. The

A

  c c e p t e d M a n u s c r i p t resultant oxacillin-resistant subpopulations can be defined as hl-MRSA populations because of the high oxacillin concentration used for selection of MRSA.

  A c c e p t e d M a n u s c r i p t 0.30, 0.40, 0.48, 0.60, 0.80, 0.96, 1.2, 1.6, 2.4, 3.2, 3.8, 4.8, 6.4, 7.7, 9.6, 12.8, 15.5, 19.2, 25.6, 31.0, 38.4, 51.0, 62.0, 76.8, 102.4 and 128 mg/L.

A

  c c e p t e d M a n u s c r i p t profiles of agents with short half-lives (e.g. amoxicillin t 1/2 = 3.5 h; ciprofloxacin t 1/2 = 5.0 h), bacteria were entrapped in dialysis tubing (neoLab, Heidelberg, Germany) fixed within the central compartment. Antibacterials selectively diffuse into the tubing whereas bacteria do not penetrate into the surrounding medium. Bacteria were not washed-out from the central compartment provided the simulated t 1/2 values exceeded 8 h (e.g. levofloxacin and moxifloxacin)[START_REF] Dalhoff | Pharmacodynamics of fluoroquinolones[END_REF].Bacteria were grown in BHI broth and were incubated in ambient air at 37 C.Viable counts were determined at 0, 0.5, 1, 2, 3, 5, 7, 12, 24, 25, 26, 27, 29, 36, 48, 49, 50, 51, 53, 55, 60, 72, 73, 74, 75, 77, 79, 84 and 96 h. The impact of fluoroquinolone or amoxicillin/clavulanic acid carry-over on viable counts was minimised first by diluting the samples and second by plating the aliquots on cation-enriched agar or Bacillus cereus -lactamse-containing agar, thus inactivating the agents. At the point of quantification of viable counts, antibiotic concentrations were measured by a conventional cup-plate agar diffusion test with Bacillus subtilis spore suspension as the indicator organism. Drug concentrations were monitored in the absence and presence of bacteria.

  Calculated free serum concentrations (amoxicillin C max = 9.5 mg/L and AUC = 44.4 mg h/L; ciprofloxacin C max = 2.0 mg/L and AUC = 16.1 mg h/L; levofloxacin C max = 4.8 mg/L and AUC = 40.5 mg h/L; and moxifloxacin C max = 2.3 and AUC = 19.8 mg h/L) simulated in the in vitro pharmacodynamic model are illustrated in Fig. 1. Viable counts were quantitated by spreading the samples onto agar plates containing B. cereus -lactamse (Merck KGaA, Darmstadt, Germany) or 5 mM calcium to inactivate amoxicillin or the fluoroquinolones. In parallel, samples were plated first onto agar plates containing 2, 4 and 8 the MIC of the fluoroquinolone studied; and second, samples were plated onto agar plates containing 2% NaCl + 32, 64 and 128 mg/L oxacillin and incubated at 35 C. Thus, the emergence of fluoroquinolone-resistant subpopulations as well as the emergence of MRSA heteroresistance is addressed in parallel in these experiments.

  -resistant subclones by fluoroquinolones has been studied by population analysis profiling. The indicator MRSA strain P8 and its laboratory-generated fluoroquinolone-resistant mutants were exposed to a broad range of fluoroquinolone concentrations and total viable counts were A c c e p t e d M a n u s c r i p t quantitated as well as the frequency with which highly meticillin-resistant subclones (128 mg/L oxacillin) emerged upon exposure to any of the fluoroquinolone concentrations (Table

  discussed above support the notion that quinolone resistance in staphylococci is not a class effect. The propensities for resistance development in vitro as well as in the clinical setting are dissociated, with ciprofloxacin and levofloxacin being better selectors of meticillin resistance and better drivers of quinolone resistance than the newer fluoroquinolones that have enhanced anti-Gram-positive activity. Thus, the increasing use of generic fluoroquinolones or inappropriately dosed levofloxacin in the treatment of community-acquired respiratory tract infections purely for economic reasons may jeopardise the beneficial clinical efficacy of the modern fluoroquinolones. Prescription guidelines should also consider the dissociated propensities for resistance development.

Funding

  s c r i p t [43] Bisognano C, Vaudaux P, Rohner P, Lew DP, Hooper DC. Inhibition of fibronectin-binding proteins and increased adhesion of quinolone-resistant Staphylococcus aureus by subinhibitory levels of ciprofloxacin. Antimicrob Agents Chemother 2000;44:1428-37.

Fig. 1 .Fig. 2 .

 12 Fig. 1. Measured drug concentrations representing the free fraction following

Fig. 3 .

 3 Fig. 3. Effect of fluctuating ciprofloxacin concentrations simulating oral doses

Fig. 4 .

 4 Fig. 4. Effect of fluctuating levofloxacin concentrations simulating oral doses

Fig. 5 .

 5 Fig. 5. Effect of fluctuating moxifloxacin concentrations simulating oral doses

2  6 CA 1 FQ 4 A c c e p t e d M a n u s c r i p t Table 2

 26142 Range of initial minimal inhibitory concentrations (MICs) (mg/L), spontaneous mutation frequency (smf) towards fluoroquinolone resistance (4 initial MIC) and selection of high-level meticillin resistance (128 mg/L oxacillin) in the presence of 0.5 the minimal inhibitory concentrations of the study drugs  10 -8 to 2.3  10 -10 3.6  10 -8 to 8.8  10 -9 1.0  10 -9 to 10 -10 -8 to 2.8  10 -9 5.2  10 -8 to 5.0  10 -9 1.0  10 -9 to 10-11 Resistance selection 1.1  10 -3 to 3.3  10 -5 2.2  10 -4 to 7.1  10 -5 3.2  10 -5 to 4.1  10 -10 -8 to 3.2  10 -9 4.8  10 -8 to 6.2  10 -9 2.2  10 -9 to 10-11 Resistance selection 4.6  10 -3 to 6.8  10 -5 5.2  10 -4 to 8.6  10 -5 N/D 10 -7 to 8.4  10 -8 2.8  10 -7 to 5.6  10 -8 6.7  10 -8 to 10-11 Resistance selection nd nd nd A c c e p t e d M a n u s c r i p t MSSA, meticillin-susceptible Staphylococcus aureus; MRSA, meticillin-resistant S. aureus; HA, healthcare-associated; CA, community-acquired; FQ r , fluoroquinolone-resistant; N/D, not done; nd, not done. a FQ r MRSA strains were generated in the laboratory by serial passage until the initial MIC has been increased eight-fold. Selection of highly meticillin-resistant (128 mg/L oxacillin) subclones of the heteroresistant meticillin-resistant Staphylococcus aureus (MRSA) strain P8 by constant fluoroquinolone concentrations. The parent strain P8, as well as the laboratory-generated mutants with 2, 4 and 8 elevated fluoroquinolone minimal inhibitory concentrations (MICs)

Competing interests

None declared.

Ethical approval

Not required.


Justin L Copitch

Rebekah N Whitehead

Mark A Webber
email: m.a.webber@bham.ac.uk

Prevalence of decreased susceptibility to triclosan in Salmonella enterica isolates from animals and humans and association with multiple drug resistance

Keywords:

A c c e p t e d M a n u s c r i p t ABSTRACT Previous laboratory studies have implicated triclosan as a possible selective force driving resistance to multiple antibiotics and have identified a number of triclosan resistance mechanisms in Salmonella enterica. The aim of this work was to determine the prevalence of decreased susceptibility to triclosan in a panel of human and animal isolates of S. enterica and to identify the mechanisms of triclosan resistance in these strains. Over 400 animal and human isolates of non-typhoidal Salmonella were screened for decreased susceptibility to triclosan and a panel of antibiotics. The prevalence of decreased susceptibility to triclosan was ca. 4%. Of the isolates with decreased triclosan susceptibility, 56% were multidrug-resistant (MDR) compared with 12% of triclosan-sensitive isolates. MDR and triclosanresistant strains showed increased efflux activity compared with strains with reduced susceptibility to triclosan alone. No high-level triclosan resistance was seen in this panel of isolates. A reservoir of strains with low-level decreased triclosan susceptibility is present in animals and humans. These isolates are MDR as a result of generic mechanisms of antimicrobial resistance and do not carry specific mutations within fabI.

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

Introduction

Non-typhoidal Salmonella (NTS) are a major cause of enteric illness both in humans and animals [START_REF] Roberts | Infectious Intestinal Disease Study Executive. The study of infectious intestinal disease in England: socio-economic impact[END_REF]. NTS usually cause a self-limiting mild illness but can have serious sequelae. The primary route of NTS infection in humans is via ingestion of contaminated food, in particular poultry and eggs [START_REF] Louis | The emergence of grade A eggs as a major source of Salmonella enteritidis infections. New implications for the control of salmonellosis[END_REF]. Antibioticresistant Salmonella strains resistant to multiple therapeutic drugs are becoming increasingly common, and infection with a multidrug-resistant (MDR) strain of Salmonella significantly increases the risk of morbidity and mortality [START_REF] Helms | Excess mortality associated with antimicrobial drug-resistant Salmonella Typhimurium[END_REF].

Triclosan is a broad-spectrum antimicrobial bisphenol biocide [START_REF] Schweizer | Triclosan: a widely used biocide and its link to antibiotics[END_REF]. It is used in a wide array of domestic products including deodorants, soaps, kitchen utensils and toothpastes [START_REF] Schweizer | Triclosan: a widely used biocide and its link to antibiotics[END_REF]. When first introduced, the mechanism of action of triclosan was unknown and was considered to be non-specific, acting to disrupt bacterial membranes [START_REF] Regös | Investigations on the mode of action of triclosan, a broad spectrum antimicrobial agent[END_REF]. In the early 1990s, triclosan-resistant Staphylococcus aureus were described, and several later studies with Escherichia coli, Pseudomonas aeruginosa, Mycobacterium smegmatis, Mycobacterium tuberculosis and S. aureus showed that triclosan does in fact have a defined bacterial target, the FabI enzyme, essential for fatty acid synthesis [START_REF] Mcmurry | Triclosan targets lipid synthesis[END_REF]. Several mechanisms of resistance to triclosan have been demonstrated in different bacteria. These include mutations in or upregulation of fabI, derepression of efflux pumps, carriage of alternative enoyl-acyl reductases, production of degradative enzymes and alteration of fatty acid metabolism pathways [START_REF] Heath | Mechanism of triclosan inhibition of bacterial fatty acid synthesis[END_REF][START_REF] Meade | Soil bacteria Pseudomonas putida and Alcaligenes xylosoxidans subsp. denitrificans inactivate triclosan in liquid and solid substrates[END_REF].

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

Of particular concern has been the demonstration of upregulation of efflux pumps in response to biocide exposure, including triclosan, a consequence of which is an increased degree of resistance to multiple drugs, albeit at a relatively low level [START_REF] Levy | Antibacterial household products: cause for concern[END_REF].

In this study, we sought to identify human and animal isolates of Salmonella enterica with decreased susceptibility to triclosan, to investigate whether these isolates were MDR and to analyse the mechanisms of triclosan resistance in isolates with reduced susceptibility.

Materials and methods

Bacterial strains

Over 400 isolates (60 human isolates and 368 animal isolates) of S. enterica (comprising 40 serovars) from animals or humans collected over the last 20 years were screened for resistance to triclosan. All bacterial strains were stored at -20 C on Protect™ beads.

Identification of triclosan-resistant Salmonella isolates

All strains were inoculated onto LB agar plates containing 0.12 mg/L triclosan, a concentration that equates to two times the minimum inhibitory concentration (MIC) of wild-type strains [START_REF] Webber | Triclosan resistance in Salmonella enterica serovar Typhimurium[END_REF]. Strains were also grown on a control agar plate without triclosan as well as an XLD agar plate to presumptively identify strains as Salmonella. All strains that were unable to grow on these plates were discarded.

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

Susceptibility testing

MICs of triclosan and a panel of antibiotics, including ciprofloxacin, chloramphenicol, tetracycline, ampicillin, kanamycin and nalidixic acid, required to inhibit growth of isolates identified in the triclosan breakpoint testing were determined at least five times using the agar dilution method according to the guidelines of the British Society for Antimicrobial Chemotherapy (BSAC) [START_REF] Andrews | Determination of minimum inhibitory concentrations[END_REF]. Interpretive breakpoints for ampicillin were according to BSAC [START_REF] Andrews | Determination of minimum inhibitory concentrations[END_REF]; the following breakpoints were used for ciprofloxacin (0.25 mg/L), tetracycline (8 mg/L), chloramphenicol (8 mg/L), kanamycin (8 mg/L) and nalidixic acid (>32 mg/L) for which BSAC do not suggest breakpoints against Salmonella. Salmonella enterica serovar Typhimurium SL1344 was used as a wild-type control for susceptibility testing. Multidrug resistance was defined as resistance to three or more separate drug classes.

All strains where susceptibility testing confirmed triclosan resistance levels to be higher than that in SL1344 were retained for further characterisation.

Genotyping of fabI

Polymerase chain reaction (PCR) was used to amplify fabI from strains of interest using specific primers (AAC GTC ACC TGC CGG AGA TA and TGG ATT ATC CTG GCG TAT GC). The resulting amplimers were sequenced at the Functional Genomics Laboratory at the University of Birmingham (Birmingham, UK). Sequences were analysed and compared with the fabI sequences from S. Typhimurium SL1344, LT2 and DT104 and from E. coli K-A c c e p t e d M a n u s c r i p t 12, CFT073, 101-1, 536 and 042 strains using the programme ClustalW2, and mutations resulting in amino acid changes were recorded. The consequences of substitutions within FabI were predicted using the SWISS-MODEL programme.

Transduction of mutations

Where mutations resulting in amino acid changes were found in fabI, P22 phage transduction was used to transfer the mutant fabI allele into SL1344 to determine the role of these mutations in resistance to triclosan [START_REF] Hartman | Some improved methods in P22 transduction[END_REF].

Transductants were recovered on LB agar plates containing 0.25 mg/L triclosan. The MIC of triclosan required to inhibit these transductants was determined and fabI was sequenced to identify any specific target site mutations.

Efflux activity

To determine the level of efflux activity of strains with decreased triclosan sensitivity, intracellular accumulation of the fluorescent dye Hoechst 33342 was measured as previously described [START_REF] Gunell | Mechanisms of resistance in non-typhoidal Salmonella enterica exhibiting a non-classical quinolone resistance phenotype[END_REF]. Hoechst 33342 intercalates with DNA and fluoresces, allowing intracellular accumulation to be identified. It is also a substrate of the major multidrug efflux system AcrAB-TolC. Accumulation was measured using a FLUOstar OPTIMA (BMG Labtech, Offenburg, Germany) in the presence and absence of the efflux pump inhibitor phenyl-arginine -naphthylamide (PA N) added to cultures to a final concentration of 100 mg/L (the MIC of this compound is >400 mg/L). Mean A c c e p t e d M a n u s c r i p t accumulation values were calculated from eight biological replicates and standard deviations were calculated. Student's t-test was used to assess the significance of differences between strains.

Expression of acrB

Expression of acrB by isolates with an active efflux phenotype was determined by reverse transcriptase PCR. RNA was prepared from duplicate biological cultures (grown to an optical density at 600 nm of ca. 0.7). Five millilitres of culture was harvested and RNA was prepared using the SV40 Total RNA Isolation Kit (Promega, Southampton, UK) according to the manufacturer's recommendations. Quantities of RNA were determined using a NanoDrop (Fisher Scientific, Loughborough, UK). Two micrograms of RNA was reverse-transcribed into cDNA using Superscript III (Invitrogen, Paisley, UK) and two replicates were made for each RNA preparation giving four samples from each strain. Specific primers were used to amplify 16S rRNA as a control (cDNA diluted 1:1000) and acrB (undiluted cDNA used as template). Amplimers (5 L) were separated on 1% agarose gels and band intensities were determined using the GeneTools program (Syngene, Cambridge, UK).

Average intensities of 16S rRNA and acrB were calculated for each strain, and expression of acrB was normalised using data from 16S rRNA.

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

Results

Prevalence of triclosan resistance

Of the 428 strains of Salmonella screened, 111 showed growth on plates containing 0.12 mg/L triclosan and appeared to be characteristic of Salmonella on XLD agar. Of these 111 strains, 16 were found to consistently have a higher MIC of triclosan than SL1344 and were selected for further characterisation. The level of resistance to triclosan in these 16 strains was low and 15/16 isolates were inhibited by 0.25 mg/L or 0.5 mg/L triclosan; one isolate (L298) demonstrated a higher level of triclosan resistance and required 4 mg/L of triclosan for inhibition (Table 1). Of these 16 strains, 9 (56%) were MDR. Full MIC profiles were also determined for 33 isolates from the same panel that did not show increased resistance to triclosan, of which only 4 (12%) were MDR. The prevalence of multidrug resistance amongst triclosanresistant strains suggests an association between multidrug resistance and triclosan resistance and was statistically significant according to the 2 test (P < 0.05).

Genotyping of fabI and significance of mutations

When sequenced, five strains with decreased triclosan susceptibility were found to carry missense mutations within fabI. Strains L308, L406, L488, L494 and L501 all possessed the same change of valine to isoleucine at codon 115. All of these strains had a low triclosan MIC (0.25 mg/L or 0.5 mg/L). No other missense mutations were found in any strain. Codon 115 is at the edge of one of the two active site regions of FabI (codons 95-115 and 190-210),

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

suggesting that substitutions here may be relevant to function. To determine whether this novel substitution has a role in triclosan resistance, a variety of approaches were used. Bioinformatic analysis of the S. Typhimurium LT2 FabI sequence using the SIFT algorithm predicted that this substitution would be well tolerated by the protein. In agreement with this, the mutant FabI allele was modelled by SWISS-MODEL, which predicted a secondary structure virtually indistinguishable to the wild-type protein (data not shown).

Furthermore, alignment of FabI amino acid sequences from four strains of Salmonella (S. Typhimurium LT2, S. Typhimurium DT104, S. Typhimurium SL1344 and S. Enteritidis PT4) and five strains of E. coli (CFT073, 536, 101-1, 042 and K-12) using ClustalW2 revealed this substitution to be present in two of the five Salmonella FabI sequences and all the E. coli sequences. Susceptibility of wild-type strains of both species to triclosan is very similar and it would appear (in conjunction with transduction results; see below) that this substitution at codon 115 does not contribute to increased triclosan resistance.

Transduction of triclosan resistance

Strain L488, which demonstrated decreased susceptibility to triclosan and contained the Val115Ile mutation, was used as a donor to transduce the mutation(s) conferring decreased triclosan susceptibility into SL1344 using P22 phage. Transductants were successfully recovered on agar plates containing 0.25 mg/L triclosan. Genotyping of FabI from these transductants revealed a wild-type sequence, demonstrating that the substitution at codon 115 of the parent did not confer the decreased triclosan susceptibility of this A c c e p t e d M a n u s c r i p t isolate as it was not transferred even though the triclosan resistance phenotype was.

Efflux activity of triclosan mutants

Accumulation of the fluorescent intercalating dye Hoechst 33342, which is a substrate of the AcrAB-TolC efflux system, was analysed for all isolates with decreased susceptibility to triclosan (Fig. 1). Strains L308, L424, L616 and L631 all accumulated <50% as much Hoechst 33342 as SL1344, and strains L292, L298, L306, L494 and L501 accumulated <25%. Six isolates (L292, L308, L325, L424, L616 and L631) showed a greater than four-fold increase in accumulation of Hoechst 33342 when an efflux pump inhibitor was added, indicating that the low levels of dye accumulation in these strains is likely to be a result of increased expression of efflux pumps. Expression of acrB was increased in all the isolates with decreased triclosan sensitivity compared with that of SL1344 (Fig. 2). This correlated well with the Hoechst accumulation data and demonstrates that the isolates with decreased triclosan susceptibility are efflux mutants that overexpress acrB.

Discussion

Recently we have described the multifactorial basis of resistance to triclosan in Salmonella and have shown that very high-level resistance to triclosan is relatively easy to select in laboratory conditions. In this study, we wanted to know whether similar phenotypes are seen in real world isolates from animals and man and whether decreased susceptibility to triclosan is associated with A c c e p t e d M a n u s c r i p t multidrug resistance. Of the 428 isolates of Salmonella that were screened for decreased susceptibility to triclosan, ca. 4% demonstrated decreased susceptibility to triclosan. Analysis of these isolates showed that the level of resistance to triclosan was generally low (triclosan MIC range 0.25-4 mg/L), suggesting that different mechanisms of resistance are present in different isolates. Decreased susceptibility to triclosan was seen in eight separate serovars, indicating no association with a particular serotype or clone. These findings are similar to a previous report by Randall et al. [START_REF] Randall | Association between cyclohexane resistance in Salmonella of different serovars and increased resistance to multiple antibiotics, disinfectants and dyes[END_REF] that found only one strain of Salmonella with a triclosan MIC > 0.5 mg/L from a panel of 388 isolates. Studies of triclosan susceptibility in oral commensal flora have found little or no evidence of triclosan resistance. Another large study of bacteria isolated from hands in a community setting found no statistically significant correlation between susceptibility to triclosan and antibiotics, although there was an association between strains with higher triclosan MICs and antibiotic resistance [START_REF] Aiello | Relationship between triclosan and susceptibilities of bacteria isolated from hands in the community[END_REF]. Whilst in agreement with previous studies in that the incidence and level of triclosan resistance in these isolates is low, of note in this study was the marked association between decreased triclosan susceptibility and multidrug resistance. Significantly higher rates of multidrug resistance were seen amongst isolates with reduced susceptibility to triclosan (56%) than among triclosan-sensitive isolates (12%).

One of the main aims of this study was to determine the mechanisms of triclosan resistance prevalent in isolates and to identify whether the diverse laboratory-selected phenotypes observed previously were present in field isolates [START_REF] Webber | Triclosan resistance in Salmonella enterica serovar Typhimurium[END_REF]. No significant contribution of fabI mutations to triclosan A c c e p t e d M a n u s c r i p t susceptibility was seen in any isolate. The only substitution observed in these isolates was predicted by bioinformatics to have little effect on FabI and was not transferred when resistance was transduced from a strain carrying the substitution to a wild-type strain. It therefore seems unlikely that FabI contributes to the low-level seen in this panel of isolates. Whilst studies with laboratory mutants commonly identify mutants resistant to triclosan with alterations in FabI, it may be that in a competitive environment alterations to FabI may carry a significant fitness cost prohibitive to their selection and expansion. The main mechanism of resistance observed was efflux. The majority of isolates with decreased resistance to triclosan were also MDR and demonstrated greater efflux activity and acrB expression than control strains. The absence of 'triclosan-specific' mechanisms of resistance as seen in our previous studies with laboratory-selected mutants with very high levels of triclosan resistance may reflect a fitness cost of these mutations or the lack of high-level triclosan selective pressure in the environment.

Whether the derepression of MDR efflux systems seen in these isolates is a consequence of triclosan exposure in the environment or other substrates is not possible to determine. However, the increasing proliferation of triclosan in the environment is a concern as the data presented here suggest that MDR efflux pump derepression is an effective mechanism allowing survival at relatively low levels of triclosan. Therefore, low levels of triclosan in the environment may promote selection of multidrug resistance via efflux pump derepression, which is a phenotype of clinical relevance. The use of triclosan should be restricted to applications where it is likely to be beneficial, and the proliferation of its use in domestic products is questionable.

Fig. 1 .

 1 Fig. 1. Accumulation of Hoechst 33342 by isolates with decreased triclosan

Fig. 2 .

 2 Fig. 2. Expression of acrB by isolates with decreased triclosan susceptibility.

Table 1

 1 Antimicrobial susceptibility profiles for triclosan-resistant Salmonella strains and controls a Drug abbreviations and breakpoints used: TRI, triclosan (0.25 mg/L); CIP, ciprofloxacin (0.25 mg/L); TET, tetracycline (8 mg/L); AMP, ampicillin (16 mg/L); CHL, chloramphenicol (8 mg/L); KAN, kanamycin (8 mg/L); NAL, nalidixic acid (>32 mg/L).

	Edited Table 1
	Strain	Serovar	Source	Year isolated MIC (mg/L)
					TRI CIP	TET AMP CHL KAN NAL
	SL1344 Typhimurium Wild-type control		0.12 0.015 2	2	4	2	4
	L702	Typhimurium Triclosan-resistant control		64	0.25 4	4	8	16	>128
	L10	Typhimurium Human	1990	0.5 0.06 4	4	8	16	8
	L292	Saintpaul	Poultry	1992	0.5 0.5	4	>32 8	4	>128
	L298	Anatum	Poultry	1992	4	0.5	2	1	4	32	>128
	L306	Typhimurium Poultry	1993	0.25 0.25 2	2	8	32	>128
	L308	Ohio	Poultry	1993	0.5 0.5	4	4	16	16	>128
	L325	Typhimurium Poultry	1993	0.5 1	>32 >32 >32 16	>128
	L361	Typhimurium Poultry	2000	0.5 0.06 4	1	>32 16	>128
	L406	Senftenberg Poultry	2000	0.25 0.5	2	2	8	32	>128
	L424	Typhimurium Poultry	2000	0.5 0.12 8	16	>32 16	16
	L476	Senftenberg Poultry	2000	0.25 0.5	2	1	4	32	>128
	L488	Newport	Poultry	2000	0.25 0.5	2	1	16	32	>128
	L494	Indiana	Poultry	2000	0.25 0.5	2	1	4	32	>128
	L501	Typhimurium Poultry	2000	0.25 0.25 2	1	4	32	>128

a

Funding

Research into triclosan is funded by a BBSRC David Phillips Fellowship (BB/D020476/1) to MAW.

Competing interests

None declared.

Ethical approval

Not required.