David J Farrell

Marion Robbins

William Rhys-Williams

William G Love

In vitro activity of XF-73, a novel antibacterial agent, against antibiotic-sensitive and -resistant Gram-positive and Gramnegative bacterial species

Keywords:

E-mail address: wl@destinypharma.com (W.G. Love). 1 Present address: JMI Laboratories, 345 Beaver Kreek Centre, North Liberty, IA 52317, USA.

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

ABSTRACT

The antibacterial activity of XF-73, a dicationic porphyrin drug, was investigated against a range of Gram-positive and Gram-negative bacteria with known antibiotic resistance profiles, including resistance to cell wall synthesis, protein synthesis, and DNA and RNA synthesis inhibitors as well as cell membrane-active antibiotics. Antibiotic-sensitive strains for each of the bacterial species tested were also included for comparison purposes. XF-73 was active [minimum inhibitory concentration (MIC) 0.25-4 mg/L] against all of the Gram-positive bacteria tested, irrespective of the antibiotic resistance profile of the isolates, suggesting that the mechanism of action of XF-73 is unique compared with all other antibiotic classes tested. Gram-negative activity was lower (MIC 1 mg/L to >64 mg/L). Minimum bactericidal concentration data confirmed that the activity of XF-73 was bactericidal. Time-kill kinetics against healthcare-associated and community-associated meticillin-resistant Staphylococcus aureus isolates demonstrated that XF-73 was rapidly bactericidal, with >5 log 10 kill obtained after 15 min at 2 MIC, the earliest time point sampled. The post antibiotic effect (PAE) for XF-73 under conditions where the PAE for vancomycin was <0.4 h was found to be >5.4 h. XF-73 represents a novel broad-spectrum Gram-positive antibacterial drug with potentially beneficial characteristics for the treatment and prevention of Gram-positive bacterial infections.

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

Introduction

The impact of antibiotic-resistant bacteria on the treatment of infections continues its relentless march upwards. Meticillin-resistant Staphylococcus aureus (MRSA) is the prime example; MRSA is increasingly prevalent throughout the world and is endemic in some hospital and community settings [START_REF]Surveillance of healthcare associated infections report[END_REF][START_REF]MRSA in healthcare settings[END_REF][START_REF] Moran | EMERGEncy ID Net Study Group. Methicillin-resistant S. aureus infections among patients in the emergency department[END_REF]. Healthcare-associated MRSA (HA-MRSA) now accounts for 40% of S. aureus infections in the UK and 63% of S. aureus infections in the USA [START_REF]Surveillance of healthcare associated infections report[END_REF][START_REF]MRSA in healthcare settings[END_REF].

Community-associated MRSA (CA-MRSA) is also becoming more widespread; in the USA, 59% of skin and soft-tissue infections (SSTIs) presenting to emergency departments are caused by CA-MRSA [START_REF] Moran | EMERGEncy ID Net Study Group. Methicillin-resistant S. aureus infections among patients in the emergency department[END_REF]. The most prevalent CA-MRSA clone in the USA, USA300, which carries the staphylococcal cassette chromosome mec (SCCmec) type IVa and Panton-Valentine leukocidin genes, now accounts for 61-87% of MRSA isolates from SSTIs in North American emergency departments [START_REF] King | Emergence of community-acquired methicillin-resistant Staphylococcus aureus USA 300 clone as the predominant cause of skin and soft-tissue infections[END_REF][START_REF] Gn | Community-associated CMRSA-10 (USA-300) is the predominant strain among methicillin-resistant Staphylococcus aureus strains causing skin and soft tissue infections in patients presenting to the emergency department of a Canadian tertiary care hospital[END_REF] and is also emerging as a serious cause of healthcare-associated infections [START_REF] Kourbatova | Emergence of community-associated methicillin-resistant Staphylococcus aureus USA 300 clone as a cause of health care-associated infections among patients with prosthetic joint infections[END_REF][START_REF] Gonzalez | Community-associated strains of methicillin-resistant Staphylococcus aureus as the cause of healthcare-associated infection[END_REF][START_REF] Seybold | Emergence of community-associated methicillin-resistant Staphylococcus aureus USA300 genotype as a major cause of health care-associated blood stream infections[END_REF].

A further complication is the emergence of multidrug-resistant (MDR) bacteria both in Gram-positive and Gram-negative species. There is a critical need for new antibacterials, particularly those with activity against MDR bacteria.

Materials and methods

Bacterial strains

A panel of 101 Gram-positive and Gram-negative organisms, including anaerobes, was selected for susceptibility and cross-resistance testing (Table 1; Supplementary Table 1). The panel comprised 65 Gram-positive isolates (56 aerobic and 9 anaerobic) and 36 Gram-negative isolates (31 aerobic and 5 anaerobic). Examples of isolates resistant to cell wall inhibitors (-lactams, glycopeptides and cephalosporins), protein synthesis inhibitors (oxazolidinones, macrolides and mupirocin), DNA synthesis inhibitors (fluoroquinolones), RNA synthesis inhibitors (rifampicin) and bacterial cell membrane-active antibiotics (daptomycin) were included in the panel.

The Gram-positive panel (Table 1 Clinical and Laboratory Standards Institute (CLSI) antimicrobial susceptibility control strains were included in the screen to confirm that the expected MICs were obtained for the comparator compounds (vancomycin, linezolid and clindamycin) and, where possible, an antibiotic-sensitive and an antibioticresistant isolate was included for all species tested.

For investigation of killing kinetics and determination of the PAE, two NARSA [START_REF]Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA)[END_REF] strains were used, comprising one CA-MRSA (NRS-123) and one HA-MRSA (NRS-382).

Antimicrobial agents

Media used

Mueller-Hinton (MH) broth (Oxoid Ltd., Basingstoke, UK) was used to prepare the inoculum and according to CLSI requirements [13,14]. For fastidious isolates, MH broth was supplemented with 2.5-5% v/v lysed horse blood. For Haemophilus influenzae, plates and inocula were prepared in Haemophilus test medium (Trek Diagnostic Systems Ltd., East Grinstead, UK). For anaerobic isolates, supplemented Brucella broth was prepared according to CLSI recommendations [13,14].

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

Determination of bactericidal activity by time-kill

XF-73, vancomycin, fusidic acid and mupirocin were added at final concentrations of 2, 4 and 8 MIC to exponentially dividing cultures of the test strains. A higher final bacterial concentration of ca. 10 6 CFU/mL was utilised to allow an ca. 5 log 10 reduction to be measured. Cultures were incubated at 37 ± 1 C in a shaking water-bath and samples were removed for determination of surviving bacteria using the spiral plating system (Don Whitley Scientific, Shipley, UK) on Iso-Sensitest agar after 0, 0.25, 0.5, 1, 3, 6 and 8 h of exposure. To remove the antimicrobials prior to performing viable counts, each time point sample was spun and washed twice with sterile Iso-Sensitest broth. A growth control was run under the same conditions. Count plates were incubated for 48 h and the kinetics of bactericidal activity was determined by plotting the concentration of surviving bacteria against time.

The absolute limit of detection was 2  10 1 CFU/mL.

Determination of the post-antibiotic effect

The PAE is the duration of bacterial growth suppression following abrupt removal of the test antibacterial from the culture medium [START_REF] Craig | The postantibiotic effect[END_REF][START_REF] Vogelman | Postantibiotic effects[END_REF][START_REF] Zhanel | The postantibiotic effect: a review of in vitro and in vivo data[END_REF]. This duration is related to the antibacterial concentration and exposure time prior to its removal [START_REF] Craig | The postantibiotic effect[END_REF] and is also specific for the antibacterial-bacterium system [START_REF] Craig | The postantibiotic effect[END_REF][START_REF] Zhanel | The postantibiotic effect: a review of in vitro and in vivo data[END_REF]. XF-73 was added at a final concentration of 0.5, 1 and 2 MIC and vancomycin at a final concentration of 2 MIC to exponentially dividing CFU/mL). Following exposure for 30 min, each culture was spun and washed twice with sterile Iso-Sensitest broth and the re-suspended culture was then incubated in a shaking water-bath at 37 ± 1 C until re-growth was visible with the naked eye or for a period of 8 h. The control culture was diluted 1:100 and 1:1000 to account for antibacterial activity that may occur in the exposed cultures in order to provide a comparable starting inoculum on re-incubation for PAE calculation. The three controls were also spun and washed before sampling all of the cultures, at time point 0. Thereafter, both control and 'exposed' cultures were counted at hourly intervals using the spiral plating method.

Count plates were incubated for 48 h and the PAE was determined by plotting the concentration of surviving bacteria against time. The PAE was calculated as the time taken for the test culture to increase by 1.0 log 10 CFU/mL minus the time taken for the control culture to increase by 1.0 log 10 CFU/mL.

Results

XF-73 spectrum of activity

Gram-positive activity

XF-73 was active (MIC range 0.25-4 mg/L) (Table 1; Supplementary Table 1) against all of the aerobic and anaerobic Gram-positive organisms tested in this study. The MBC for all isolates was achieved at, or for some species A c c e p t e d M a n u s c r i p t 11 within, 1-2 dilutions of the MIC (Supplementary Table 1), demonstrating that the antibacterial activity was bactericidal. MICs of the comparator antibiotics were as expected for the CLSI antimicrobial susceptibility control strains (Table 1; Supplementary Table 1).

Gram-negative activity

MICs for XF-73 against the Gram-negative species tested ranged from 1 mg/L to >64 mg/L (Table 1; Supplementary Table 1). MICs of the comparator antibiotics were as expected for the CLSI antimicrobial susceptibility control strains (Table 1; Supplementary Table 1).

Activity of XF-73 against antibiotic-susceptible and antibiotic-resistant isolates

The antimicrobial activity of XF-73 was independent of the antibiotic resistance status for the Gram-positive bacteria tested. In every case for XF-73, the MICs for antibiotic-resistant strains were similar or identical to those observed for the antibiotic-susceptible strains. XF-73 was active against all 12 of the MRSA clones tested, with MICs in the range 0.5 mg/L to 1 mg/L, the same as that for the 3 meticillin-sensitive isolates tested. The presence of resistance genes against cell wall synthesis inhibitors, protein synthesis inhibitors, RNA and DNA synthesis inhibitors and cell membrane-active antibiotics in the MRSA strains screened also had no effect on the MICs (Table 1; Supplementary Table 1). The range of MICs for XF-73 against penicillin-resistant or macrolide-resistant Streptococcus spp. was 0.25-4 mg/L A c c e p t e d M a n u s c r i p t 12 and for susceptible isolates was 0.5-2 mg/L. XF-73 demonstrated a similar order of activity against vancomycin-resistant enterococci, where the MIC range was 0.5-2 mg/L, compared with 1-2 mg/L for susceptible isolates.

Determination of bactericidal activity by time-kill

The in vitro bactericidal activity of XF-73, vancomycin, fusidic acid and mupirocin at 2, 4 and 8 MIC against S. aureus NRS-382, an HA-MRSA strain, and NRS-123, a CA-MRSA strain, was investigated and the results for 2 MIC are shown in Fig. 1. The results demonstrated a rapid bactericidal activity, with a >5 log 10 reduction obtained even at 2 MIC after the first sampling point (15 min). This compares with a reduction of <1 log 10 for the three comparators. There was an increase in CFUs between 6 h and 8 h for XF-73 at 2 and 4 MIC, but this was due to the higher initial inoculum used for this study. At 8 MIC, no increase in CFUs was observed between 6 h and 8 h (data not shown).

Post-antibiotic effect

Owing to the rapid bactericidal activity of XF-73, the PAE was determined at 0.5, 1 and 2 MIC with a relatively short exposure time of 30 min, which is one-half the usual 1 h exposure time (Fig. 2). To enable comparison using these conditions, the PAE of vancomycin at 2 MIC was also determined. For NRS-382 at 0.5, 1 and 2 MIC, a PAE of 3.5, 5 and 5.4 h, respectively, was observed. By comparison, the PAE of vancomycin at 2 MIC was significantly A c c e p t e d M a n u s c r i p t 13 shorter at 0.4 h (Fig. 2a). For NRS-123, a CA-MRSA strain, the PAE for XF-73 was longer at all three multiples tested (Fig. 2b), being recorded at 5.4, 6.1 and 6.3 h at 0.5, 1 and 2 MIC, respectively. Again, by comparison, the PAE of vancomycin at 2 MIC was 0.3 h.

Discussion

Despite the increasing threat of antibiotic drug resistance, very few new antibacterials are emerging and even fewer with novel mechanisms of action.

For instance, in the 5-year period between 2002 and 2007, only five new antibiotics were approved by the US Food and Drug Administration (FDA) [START_REF] Stephens | The war on superbugs[END_REF]. Thus, a novel bactericidal agent with potent activity against antibioticresistant strains is an important advance. XF-73 demonstrated excellent bactericidal activity against all Gram-positive strains tested regardless of species, phenotype or genotype, with MICs of 0.25-4 mg/L. The antibacterial activity against Gram-negative species was lower, with MICs of 1 mg/L to >64 mg/L.

The difference in susceptibility to some antimicrobial agents between Grampositive and Gram-negative bacteria has been attributed to the low permeability of the Gram-negative bacterial outer cell wall to antibacterial compounds [START_REF] Livermore | Antibiotic uptake and transport by bacteria[END_REF]. The bactericidal activity of XF-73 appears to involve accumulation of the molecule at, and perturbation of, the cytoplasmic membrane barrier, causing rapid cell death within 5 min [START_REF] Ooi | XF-73, a novel antistaphylococcal membrane-active agent with rapid bactericidal activity[END_REF]. The less VISA and high-level mupirocin-resistant (MIC > 256 mg/L) and daptomycinnon-susceptible (MIC = 2 mg/L) isolates. In all cases, MICs and MBCs for XF-73 were within 1 dilution multiple of one another. Results for the daptomycinnon-susceptible isolate are particularly interesting, as daptomycin, like XF-73 [START_REF] Ooi | XF-73, a novel antistaphylococcal membrane-active agent with rapid bactericidal activity[END_REF] is postulated to act primarily by disruption of the cytoplasmic membrane [START_REF] Silverman | Correlation of daptomycin bactericidal activity and membrane depolarization in Staphylococcus aureus[END_REF]. The results for XF-73 suggest that the mechanism of action of XF-73 is distinct from that of daptomycin. Macrolide-resistant isolates of Streptococcus (Group C, Group G, S. agalactiae, S. bovis, S. constellatus, S. mitis, S. oralis, S. pyogenes and S. sanguinis) all had MIC/MBCs identical to antibiotic-susceptible isolates of the same strains, suggesting that the mechanism of action of XF-73 does not involve inhibition of bacterial protein biosynthesis. This is supported by similar MICs for a high-level gentamicin-resistant isolate of Enterococcus faecalis and a CLSI antimicrobial susceptibility control strain. Identical MICs were also obtained for doxycycline-sensitive and -resistant isolates of Corynebacterium jeikeium and S. aureus as well as tetracycline-sensitive and -resistant isolates of E. faecalis and Streptococcus pneumoniae, further suggesting that inhibition of protein synthesis is not the mechanism of action of XF-73.

A number of S. aureus isolates, an isolate of S. epidermidis and Enterococcus faecium were resistant to levofloxacin. The MICs obtained for these isolates were identical to the MICs obtained for corresponding levofloxacin-sensitive isolates, suggesting that the mechanism of action of XF-73 does not involve inhibition of DNA supercoiling.

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

The bacterial time-kill experiments demonstrated that XF-73 exerted a rapid and significant bacterial kill, with a >5 log 10 reduction in viable cell numbers after only 15 min incubation time (Fig. 1), the earliest sampling time point, based on conventional experimental protocols. Bacterial cell numbers were reduced to or below the limit of detection of the assay by this first sampling point. Thus, the actual speed of bacterial kill cannot be stated. Further work needs to be undertaken to obtain data points at even earlier time points to determine what the reduction in viable cell numbers is to obtain a more accurate measure of the rate of bacterial kill.

A long PAE was obtained for XF-73 against both a CA-MRSA strain (5.4, 6.1 and 6.3 h at 0.5, 1 and 2 MIC, respectively) and an HA-MRSA strain (3.5, 5 and 5.4 h at 0.5, 1 and 2 MIC, respectively) using an exposure time that was half the standard exposure time (Fig. 2). XF-73 was found to have a PAE 21 times longer than vancomycin for NRS-123 (0.3 h at 2 MIC) and 14 times longer for NRS-382 (0.4 h at 2 MIC) when tested under the same conditions.

A long PAE is beneficial as it provides additional time for the immune system to remove bacteria that might have survived antibiotic treatment before they could eventually re-grow after removal of the drug. A longer PAE can therefore influence the clinical outcome of antimicrobial therapy.

The data presented here, along with previous studies on its antibacterial activity (including remote resistance profile [START_REF] Rhys-Williams | XF-73: a novel antimicrobial-investigation of the change in MIC against multiple MRSA strains during a 55-passage study[END_REF][START_REF] Love | XF-73: a new antimicrobial drug-comparison with daptomycin for the[END_REF] and recently confirmed o Toxin-positive and clinical isolates.

p Reference control strain and clinical isolate.

q CLSI antimicrobial susceptibility control strain.

r Erythromycin-resistant strain and clinical isolate.

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

 2 Corresponding author. Tel.: +44 1273 704 440; fax: +44 1273 704 414.

  XF-73 is a dicationic porphyrin drug and previous studies have shown that XF-73 has potent bactericidal activity [minimum bactericidal concentration for 90% of organisms (MBC 90 ) = 0.5 mg/L] in vitro against a range of clinically A c c e p t e d M a n u s c r i p t 5 important S. aureus isolates, including meticillin-sensitive S. aureus (MSSA), HA-MRSA and CA-MRSA [9]. The drug is being developed to treat S. aureus and MRSA infections, and clinical trials are underway to assess the potential of XF-73 for nasal decolonisation of S. aureus. Investigations into the mechanism of action of XF-73 against S. aureus have demonstrated that the drug exhibits a rapid cell membrane-perturbing activity that is likely to be responsible for inhibition of macromolecular synthesis and death of staphylococci exposed to the drug [10]. During prolonged laboratory subculture (55 passages) in the presence of sub-minimum inhibitory concentrations (sub-MICs) of XF-73, no resistant mutants were generated using five Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) [11] strains of MRSA [9,12]. The purpose of this study was (i) to examine the breadth of in vitro activity of XF-73, using broth microdilution, against a range of antibiotic-sensitive andresistant Gram-positive and Gram-negative aerobic and anaerobic bacterial species and (ii) to investigate the effect of existing antibiotic resistance mechanisms on the activity of XF-73. Strains in the screening panel were chosen to include examples of bacteria with all known antibacterial resistance mechanisms. Comparator antibiotics were included to confirm the expected MICs for reference strains and to confirm that that the MICs for clinical isolates were within the expected ranges, confirming the relevance of selection of these clinical isolates. Time-kill kinetics using S. aureus were also investigated to determine the speed of the bactericidal activity of XF-73. Determination of the post-antibiotic effect (PAE) for S. aureus of XF-73 for a A c c e p t e d M a n u s c r i p t 6 range of different multiples of the MIC was also undertaken and compared with a comparator antibiotic.

  XF-73 was obtained from Destiny Pharma Ltd. (Brighton, UK). Vancomycin (batch number 016K1102) and fusidic acid (batch number 065K1404) were obtained from Sigma-Aldrich Ltd. (Gillingham, UK), and mupirocin (batch number B143832) was obtained from Mast Diagnostics (Bootle, UK).

A c c e p t e d M a n u s c r i p t 8 2. 3 .

 83 Susceptibility testingMICs were determined by the broth microdilution method according to approved M7-A7 (aerobes) and M11-A7 (anaerobes) standards of the CLSI[13,14]. MIC values were read after 24 h or 48 h of incubation, depending on the species. MBC values were determined by subculturing wells showing no visible growth onto pre-labelled and quartered Columbia blood agar plates. Plates were incubated appropriate to the species and the number of surviving bacterial colonies was counted. MBC values were determined for each isolate as the antimicrobial concentration producing a 3 log 10 drop (99.9%) in bacterial colony-forming units (CFU)/mL compared with the original inoculum, based on a 5% error for determination of final inoculum[15].

  A c c e p t e d M a n u s c r i p t 10 cultures of the test strains in Iso-Sensitest broth (final concentration ca. 10 6

A

  c c e p t e d M a n u s c r i p t 14 permeable outer cell wall of Gram-negative bacteria may impede accumulation at the cytoplasmic membrane, resulting in a lower killing efficacy. Further studies are underway to characterise fully the antibacterial mechanism of action of XF-73. MICs of ≤2 mg/L for XF-73 were, however, obtained for the Gram-negative anaerobes Porphyromonas asaccharolytica and Prevotella intermedia as well as the Gram-negative aerobe Neisseria meningitidis. Moreover, the XF-73 MICs against a -lactamase-positive clinical isolate and a reduced fluoroquinolone susceptibility clinical isolate of Moraxella catarrhalis were also ≤4 mg/L, demonstrating that XF-73 did have some activity against certain Gram-negative bacteria. The strains in this screening panel were chosen to include examples of bacteria with all known antibacterial resistance mechanisms. For the S. aureus strains tested, the panel included MSSA, MRSA (SCCmec types 1-4),

A

  c c e p t e d M a n u s c r i p t 15 The bactericidal activity of XF-73 was not affected by the presence of VanA-, VanB-or VanC-derived vancomycin resistance in the enterococci isolates tested, suggesting that the mechanism of action of XF-73 does not involve inhibition of peptidoglycan synthesis. Similarly, resistance to penicillins, cephalosporins and carbapenems in other Gram-positive isolates tested did not affect the MICs obtained, suggesting that the mechanism of action of XF-73 does not involve inhibition of cell wall synthesis.

A

  c c e p t e d M a n u s c r i p t 17 bacterial biofilm activity[START_REF] Ooi | XF-70 and XF-73, novel antibacterial agents active against slow-growing and non-dividing cultures of Staphylococcus aureus including biofilms[END_REF]), suggest that XF-73 is potentially of value for the prevention and treatment of antibiotic-resistant bacterial infections. Clinical trials are now underway to assess the potential of XF-73 for nasal decolonisation of S. aureus.

A c c e p t e d M a n u s c r i p t 22 Fig. 1 .

 221 Fig. 1. In vitro bactericidal activity of XF-73 and comparators at 2 MIC

Fig. 2 .

 2 Fig. 2. Post-antibiotic effect (PAE) of XF-73 at 0.5, 1 and 2 MIC and

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

 4 Meticillin-resistant. h Vancomycin-intermediate-resistant. i Daptomycin-non-susceptible. j High-level mupirocin-resistant (MIC > 256 mg/L) clinical isolate. k Antibiotic-susceptible; meticillin-resistant and daptomycin-non-susceptible (MIC = 2 mg/L) clinical isolates. l Antibiotic-susceptible and macrolide-resistant clinical isolates. m Penicillin-susceptible, -intermediate and -resistant and MDR clinical isolates; CLSI antimicrobial susceptibility control strain. n Macrolide-resistant (MLS and M-type) and antibiotic-susceptible clinical isolates.

s

  MDR clinical isolate; CLSI -lactamase-positive control strain and CLSI antimicrobial susceptibility control strain. t -Lactamase-negative ampicillin-resistant clinical isolate and -lactamasenegative and -positive clinical isolates. u CLSI antimicrobial susceptibility control strain and MDR clinical isolate. v -Lactamase-positive and reduced fluoroquinolone susceptibility clinical isolates.

  Enterococcus spp.; and toxin-positive and ceftriaxone-resistant strains of Clostridium difficile.

	and vanC) The Gram-negative panel (Table 1; Supplementary Table 1) included MDR
	strains of Escherichia coli, Pseudomonas aeruginosa and Klebsiella
	pneumoniae.	M a n u s c r i p t
	; Supplementary Table 1) included: MRSA (control and clinical isolates, including a linezolid-resistant isolate); MSSA; vancomycin-intermediate-resistant S. aureus (VISA); high-level mupirocin-resistant (MIC > 256 mg/L) and daptomycin-non-susceptible (MIC = 2 mg/L) A c c e p t e d
	clones of S. aureus; antibiotic-susceptible and meticillin-resistant
	Staphylococcus epidermidis; antibiotic-susceptible and penicillin-and/or
	macrolide-resistant Streptococcus spp.; vancomycin-resistant (vanA, vanB
		7

Table 1

 1 In vitro activity of XF-73 and comparator antibiotics against Gram-positive and Vancomycin-susceptible and -resistant (VanA and VanB) clinical isolates.

	Edited Table 1
	Gram-negative aerobes and anaerobes
	Species (no. of isolates)	MIC (mg/L)
		XF-73 Vancomycin Linezolid Clindamycin
	Corynebacterium jeikeium	0.25	0.5-1	0.25-1	>64
	(2) a
	Enterococcus faecalis (5) b	0.5-2 0.5 to >64	1-2	16 to >64
	Enterococcus faecium (3) c	1	1 to >64	1-4	>64
	Enterococcus gallinarum (1)	1	8	2	>64
	d
	Listeria monocytogenes (1) e 0.5	0.5	2	1
	Meticillin-sensitive	0.5	0.5	2-4	0.12
	Staphylococcus aureus (2) f
	Meticillin-resistant S. aureus	0.5-1 0.5-2	1-64	0.12 to >64
	(10) g
	Vancomycin-intermediate-	1	4	2	>64
	resistant S. aureus (1) h
	Daptomycin-non-susceptible	1	NT	2	NT
	S. aureus (1) i
	High-level mupirocin-	1	NT	2	NT
	resistant S. aureus (1) j
	Staphylococcus epidermidis	0.25-	0.5	0.5-1	0.12-0.25
	(3) k	1
	Staphylococcus	0.5	4	4	0.12
	haemolyticus (1) e
	Staphylococcus	0.5	0.5	2	0.12
	saprophyticus (1) e
	Group C Streptococcus (2) l	0.5-2 0.5	1	<0.03-0.06
	Group G Streptococcus (2) l	0.25-	0.5	1	0.06
		0.5

a Antibiotic-susceptible and MDR clinical isolate. b CLSI antimicrobial susceptibility control strain; vancomycin-susceptible, vancomycin-resistant (VanA and VanB) and high-level gentamicin-resistant clinical isolates. c d Vancomycin-resistant (VanC) clinical isolate. e Clinical isolate f Meticillin-sensitive.

Acknowledgments

This work was presented in part at the 18th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), 19-22 April 2008, Barcelona, Spain (Rhys-Williams W, Love W, Hayter I, et al., abstract P559). The NARSA strains were obtained through the Network of Antimicrobial Resistance in Staphylococcus aureus (NARSA) program supported under NIAD/NIH Contract #HHSN272200700055C.

Funding

This work was supported by Destiny Pharma Ltd. (Brighton, UK).

Competing interests

DJF is a member of the scientific advisory board of Destiny Pharma Ltd. WR-W and WGL are employees of Destiny Pharma Ltd.

Ethical approval


Sara N Richter
email: sara.richter@unipd.it

Ilaria Frasson

Cristina Bergo

Riccardo Manganelli

Antonietta Cavallaro

Giorgio Palù

Characterisation of qnr plasmid-mediated quinolone resistance in Enterobacteriaceae from Italy: association of the qnrB19 allele with the integron

Keywords:

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

Introduction

Fluoroquinolones are among the most commonly prescribed antimicrobials because of their broad-spectrum antimicrobial activity. However, this intense usage of fluoroquinolones has boosted the emergence of resistance worldwide, especially in Gram-negative bacterial species. Classic quinolone resistance in Enterobacteriaceae is mediated by chromosomally encoded point mutations in the quinolone resistance-determining regions (QRDRs) of the DNA gyrase and topoisomerase IV genes or by mutation of genes regulating the expression of efflux pumps [START_REF] Hooper | Mechanisms of action and resistance of older and newer fluoroquinolones[END_REF]. More recently, plasmid-mediated quinolone resistance has been reported, with three known mechanisms to date: gyrase and topoisomerase IV protection encoded by the qnr genes [START_REF] Tran | Mechanism of plasmid-mediated quinolone resistance[END_REF][START_REF] Strahilevitz | Plasmid-mediated quinolone resistance: a multifaceted threat[END_REF]; enzymatic drug detoxification encoded by aac(6')-Ib-cr [START_REF] Robicsek | Fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase[END_REF]; and specific efflux mediated by the qepA product [START_REF] Perichon | Transferable resistance to aminoglycosides by methylation of G1405 in 16S rRNA and to hydrophilic fluoroquinolones by QepA-mediated efflux in Escherichia coli[END_REF]. The latter two mechanisms affect only fluoroquinolones with specific features (i.e. hydrophilicity and free piperazinyl amine, e.g. ciprofloxacin and norfloxacin) and hence treatment with other compounds of the same antimicrobial class (e.g. levofloxacin and ofloxacin) remains an option. In contrast, qnrmediated resistance is effective against all fluoroquinolones and hence requires treatment shift to a different class of antimicrobials. The observation that qnr is often found in association with genes that confer resistance to other classes of antibiotics [START_REF] Robicsek | The worldwide emergence of plasmid-mediated quinolone resistance[END_REF] (e.g. -lactams and aminoglycosides) implies that Enterobacteriaceae strains presenting qnr resistance may represents a serious concern in the clinical environment.

Three major groups of Qnr determinants have been described (QnrA, QnrB and QnrS), which share between 40% and 60% similarity [START_REF] Strahilevitz | Plasmid-mediated quinolone resistance: a multifaceted threat[END_REF]. More recently, two other groups have been described, qnrC [START_REF] Wang | New plasmid-mediated quinolone resistance gene, qnrC, found in a clinical isolate of Proteus mirabilis[END_REF] and qnrD [START_REF] Cavaco | qnrD, a novel gene conferring transferable quinolone resistance in Salmonella enterica serovar Kentucky and Bovismorbificans strains of human origin[END_REF].

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

In this paper, the dissemination of qnr plasmid-mediated quinolone resistance was assayed among clinical Enterobacteriaceae isolates from Italy. qnr genes were found mainly in Klebsiella pneumoniae and to a lesser extent in Escherichia coli. Among the qnrA1, qnrS1 and qnrB19 alleles found, the latter was by far the most frequent. Genetic environment analysis revealed that one qnrB19 gene was embedded in an ISCR1 complex class 1 integron, whilst all other qnrB19 genes were flanked by an ISEcp1C region as part of the Tn2012 transposon.

Materials and methods

Sample selection

In total, 232 non-duplicate clinical isolates recovered between November 2007 and May 2008 at Padua University Hospital (Italy) were selected on the basis of ciprofloxacin resistance [minimum inhibitory concentration (MIC) ≥0.25 g/mL] or extended-spectrum -lactamase (ESBL) production. In addition, ten samples fully susceptible to ciprofloxacin (MIC < 0.25 g/mL) and lacking ESBL production were also included in the study. Bacterial isolates included 157 E. coli, 47 K. pneumoniae, 2 Klebsiella oxytoca, 12 Proteus mirabilis, 5

Enterobacter aerogenes, 4 Enterobacter cloacae, 4 Citrobacter freundii and 1 Citrobacter koseri. Sample origin was mostly urine (175 samples); the remaining were from the respiratory tract [START_REF] Perez-Perez | Detection of plasmid-mediated AmpC -lactamase genes in clinical isolates by using multiplex PCR[END_REF], blood [START_REF] Cattoir | Multiplex PCR for detection of plasmid-mediated quinolone resistance qnr genes in ESBL-producing enterobacterial isolates[END_REF], skin/wounds [START_REF] Cesaro | Low selection of topoisomerase mutants from strains of Escherichia coli harbouring plasmid-borne qnr genes[END_REF], stools (2), pus/viscera [START_REF]Performance standards for antimicrobial susceptibility testing. Nineteenth informational supplement[END_REF] and the urogenital tract [START_REF] Strahilevitz | Plasmid-mediated quinolone resistance: a multifaceted threat[END_REF]. Of samples were collected, 92% were from inpatients. Patients were equally distributed between females (48%) and males (52%).

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

Antimicrobial susceptibility testing

Exact MIC values were determined by the disk diffusion test according to Clinical and Laboratory Standards Institute (CLSI) methods [START_REF]Performance standards for antimicrobial disk susceptibility tests[END_REF] and were confirmed by means of Etest strips (AB BIODISK, Solna, Sweden). ESBL production was detected according to CLSI guidelines M2-A9 and M100-S19 [START_REF]Performance standards for antimicrobial disk susceptibility tests[END_REF][START_REF]Performance standards for antimicrobial susceptibility testing. Nineteenth informational supplement[END_REF].

Polymerase chain reaction (PCR) amplification and DNA sequencing

Screening of qnrA, qnrB and qnrS genes was carried out by multiplex PCR amplification [START_REF] Cattoir | Multiplex PCR for detection of plasmid-mediated quinolone resistance qnr genes in ESBL-producing enterobacterial isolates[END_REF].

qnrC and qnrD genes were not included in this study as they were not known when the study was planned. Positive controls were represented by DNA of known qnr-positive strains: E. cloacae HM04-477 (qnrA1) [START_REF] Cambau | Occurrence of qnrA-positive clinical isolates in French teaching hospitals during 2002-2005[END_REF]; E. cloacae HM05-186 (qnrB4) [START_REF] Merens | The pentapeptide repeat proteins MfpAMt and QnrB4 exhibit opposite effects on DNA gyrase catalytic reactions and on the ternary gyrase-DNA-quinolone complex[END_REF]; and E. coli HM5-184 (qnrS1) [START_REF] Cesaro | Low selection of topoisomerase mutants from strains of Escherichia coli harbouring plasmid-borne qnr genes[END_REF].

Primers for detection of aac(6')-Ib and qepA were: aacF, 5'-ATGACTGAGCATGACCTTG-3'; aacR, 5'-AACCATGTACACGGCTGG-3'; qepAF, 5'-TGGTCTACGCCATGGACCTCA-3'; and qepAR, 5'-TGAATTCGGACACCGTCTCCG-3'.

The presence of -lactamases genes (ESBLs bla SHV , bla TEM , bla CTX-M-1 , bla CTX-M-2 , bla CTX-M-8 and bla CTX-M-9 and AmpC enzymes bla MOX-1 , bla MOX-2 , bla CMY-1-11, bla LAT-1-4 , bla BIL-1 , bla DHA-1 , bla DHA-2 , bla ACC , bla MIR-1T , bla ACT-1 , and bla FOX-1-5b ) was assessed on the qnr-positive isolates using specific primers [START_REF] Lavilla | Prevalence of qnr genes among extended-spectrum -lactamase-producing enterobacterial isolates in Barcelona, Spain[END_REF][START_REF] Li | Detection of five novel CTX-M-type extended spectrum -lactamases with one to three CTX-M-14 point mutations in isolates from Hefei, Anhui province, China[END_REF][START_REF] Perez-Perez | Detection of plasmid-mediated AmpC -lactamase genes in clinical isolates by using multiplex PCR[END_REF][START_REF] Villegas | CTX-M-12 lactamase in a Klebsiella pneumoniae clinical isolate in Colombia[END_REF].

A c c e p t e d M a n u s c r i p t PCR amplification and sequencing of the QRDR of gyrA and parC genes was performed using universal primers [START_REF] Lascols | Type II topoisomerase mutations in clinical isolates of Enterobacter cloacae and other enterobacterial species harbouring the qnrA gene[END_REF].

Primers for sequencing the genetic environment of qnrB19 genes were specific for the ISEcp1 insertion sequence or 5'-conserved sequence (5'CS) (prF ISEcp1, 5'-CGACATGGCGGTGGGTCATC-3'; prR ISEcp1, 5'-CTGCTTGTCACACATTGTAAC-3'; and pr5'CS, 5'-GGCATCCAAGCAGCAAG-3' [START_REF] Quiroga | Complex class 1 integrons with diverse variable regions, including aac(6')-Ib-cr, and a novel allele, qnrB10, associated with ISCR1 in clinical enterobacterial isolates from Argentina[END_REF]) or were specific and outward facing for the qnrB19 gene (pr5'qnrB19, 5'-GCGAGTCCAGCCGTGGAC-3'; and pr3'qnrB19comp, 5'-

CCGCAATTCAATCTGTCGATGG-3').

Positive results were confirmed by direct sequencing of PCR products.

Plasmids isolation and analysis

Plasmids from qnr-positive bacterial isolates were purified according to the Kieser protocol [START_REF] Kieser | Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli[END_REF]. Purified plasmids were run on a 0.7% Mb agarose gels at 4 C for 5 h at 4.5 V/cm and were stained with ethidium bromide.

Conjugation and transformation

Conjugation experiments were performed by a filter mating assay using a kanamycin-resistant E. coli J53 AzR strain as the recipient and selecting on Luria-Bertani (LB) agar supplemented with 100 g/mL sodium azide, 50 g/mL kanamycin and 30 g/mL nalidixic acid.

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

For transformation experiments, plasmids purified and separated on agarose gels were electroporated into E. coli TOP10 (Invitrogen Ltd., Paisley, UK) using a Bio-Rad Gene Pulser ® (Bio-Rad Laboratories, Milan, Italy). Transformants were selected on LB agar supplemented with 30 g/mL nalidixic acid.

Molecular typing

The clonal relationship among strains was assessed based on the repetitive element PCR fingerprinting method, using the 'enterobacterial repetitive intergenic consensus' (ERIC) primers ERIC1 and ERIC2 [START_REF] Lavilla | Prevalence of qnr genes among extended-spectrum -lactamase-producing enterobacterial isolates in Barcelona, Spain[END_REF].

Results

Phenotypic characterisation of isolates

qnr genes were detected in 40 (17%) of 232 unique clinical isolates of Enterobacteriaceae collected from Padua University Hospital between November 2007 and May 2008 (Table 1), comprising 32 (68%) of 47 K. pneumoniae and 8 (5.1%) of 157 E. coli. In particular, qnrA and qnrB were detected in 1 (2.1%) and 31 (66%) of 47 K. pneumoniae isolates, respectively, and qnrB and qnrS were detected in 5 (3.2%) and 3 (1.9%) of 157 E. coli isolates, respectively. No qnr gene was identified in other bacterial species and no isolate carried more than one qnr gene. All qnrB genes were identified as qnrB19 alleles [START_REF] Cattoir | ISEcp1-mediated transposition of qnrB-like gene in Escherichia coli[END_REF] by sequencing; qnrA and qnrS genes were identified as qnrA1 and qnrS1, respectively. The genes aac(6')-Ib-cr and qepA were absent in all strains with the exception of five qnrB19-positive isolates that harboured aac(6')-Ib (samples #111, #137, #143, #144 and #176; Table 1).

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

Among the 40 qnr-positive isolates, 31/40 (78%), 3/40 (7.5%), 5/40 (13%), 3/28 (11%) and 3/40 (7.5%) isolates were nalidixic acid-, ciprofloxacin-, ofloxacin-, levofloxacin-and moxifloxacin-resistant, respectively, according to CLSI criteria (Table 2). However, when considering European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, the isolates were in general more resistant to fluoroquinolones (82%, 37%, 92%, 18% and 82%, respectively).

Mutations conferring quinolone resistance

Twenty-eight isolates were identified with a D87G GyrA mutation, one isolate with two point mutations in GyrA (S83L and D87N) and one mutation in ParC (S80I) (#9), and one isolate with S83I and S80I mutations in GyrA and ParC, respectively (#70). It is interesting to note that these latter two isolates were the only qnr-positive strains resistant to all tested fluoroquinolones. On the other hand, strains showing no mutation at the topoisomerase II level (i.e. the three qnrS1-positive E. coli strains #114, #191 and # 282 and the qnrB19positive E. coli #47, #50, #60 and #132 and K. pneumoniae #97, #204 and #229) were susceptible to all tested fluoroquinolones as well as to nalidixic acid.

Association between qnr and extended-spectrum -lactamase

Since qnr genes have been associated with plasmid-mediated -lactamases [START_REF] Nordmann | Emergence of plasmid-mediated resistance to quinolones in Enterobacteriaceae[END_REF], the presence of -lactamases was assessed. In total, 30 of 40 samples contained bla genes (association of up to three different types was found in single isolates) (Table 1). Conversely, no sample carried plasmid-mediated AmpC -lactamases. ESBL production was also Finally, qnr-positive isolates remained mostly susceptible to amikacin (80%), gentamicin (85%), tetracycline (86%), trimethoprim/sulfamethoxazole (90%) and imipenem (100%), indicating no significant increase in resistance to aminoglycosides, tetracyclines, cotrimoxazole or carbapenems associated with qnr genes. The qnrA1-positive sample was the only isolate displaying simultaneous resistance to aminoglycosides, tetracyclines and cotrimoxazole; in addition, qnrS1-positive E. coli isolate #282 was resistant to tetracyclines and co-trimoxazole (data not shown).

Characterisation of the genetic environment of qnr

Analysing the plasmid electrophoretic profiles of qnr-positive isolates, all qnrB19-positive isolates showed identical profiles, with the exception of qnrB19-positive E. coli isolate #132.

The plasmid pattern of qnrA1-and qnrS1-positive isolates was similar, but differed from both qnrB19 patterns (Fig. 1).

To determine whether the qnr determinants were plasmid-encoded, qnr-containing electrophoretic bands from seven arbitrarily chosen samples (one qnrA1-, four qnrB19-and two qnrS1-positive samples) were successfully transformed into E. coli TOP10 electrocompetent cells, indicating a plasmid location for all tested qnr genes. The qnrcontaining plasmid size in each case was >23 Kb. MICs of different fluoroquinolone drugs A c c e p t e d M a n u s c r i p t against transformant strains were measured and were compared with those of the acceptor strain (i.e. E. coli TOP10). The MICs of ciprofloxacin and levofloxacin increased 27-80-fold and 80-115-fold, respectively, in the qnr-transformed strains. The MIC of nalidixic acid was the most variable, increasing from 16-fold to 128-fold in the transformant strains. When comparing transformant versus parent strains, MICs of the transformant strains decreased by at least 40-fold or 1-3-fold when the MIC of the parent strain was >1 g/mL (i.e. strains #70, #71, #111 and #146) or <1 g/mL (strains #132, #191 and #282), respectively (Table 1). In addition, bla genes were never co-transformed with qnr, indicating a different genetic location.

To determine whether the qnr-harbouring plasmid was conjugative, two qnr-positive E. coli isolates [#132 (qnrB19) and #191 (qnrS1)] were successfully used as donors in conjugation experiments. The MICs of ciprofloxacin, levofloxacin and nalidixic acid in the transconjugant strains were 14-20-, 7-and 5-10-fold higher, respectively, than those of the wild-type recipient, and 1-2-fold lower compared with those in the donor strains. Both transformed strains and transconjugants displayed no mutation at the gyrA and parC level.

The environment surrounding the qnrB19 genes was first investigated using primers complementary to the ISEcp1C region of transposon Tn2012, previously reported to be associated with the qnrB19 gene [START_REF] Cattoir | ISEcp1-mediated transposition of qnrB-like gene in Escherichia coli[END_REF]. Of 36 qnrB19-positive samples, 35 contained the ISEcp1C transposase at the 3' end of the qnrB19 gene. Sequence analysis confirmed that these qnrB19 genes were all part of the Tn2012 transposon, with the qnrB19 gene followed by the ISEcp1C element. Interestingly, these 35 isolates reported the exact same plasmid electrophoretic profile (see Fig. 1).

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

To assess the genetic environment of the remaining qnrB19-positive E. coli strain (#132), plasmid DNA was purified and subjected to direct sequencing. Analysis of a 1.3 kb qnrcontaining DNA region indicated that the qnrB19 gene was embedded in an ISCR1-containing mobile element integron complex class I. The integron was flanked by the 5'CS followed by the inverted repeat region 2 (IRR2). A truncated psp operon was immediately upstream of the qnrB19 gene. Downstream, an Int1 element with its putative recombination site was present, followed by the sapA gene disrupted by recombination events and a second recombination site (GenBank accession no. GU074393) (Fig. 2).

Clonal relationships among isolates

Finally, the clonal relationship among the isolates in this study was assessed by studying the ERIC-PCR genomic DNA profiles. As shown in Table 1, qnrB19-positive samples were grouped in seven clonally related subgroups. In addition, 17 (47%) and 8 (22%) of 36 qnrB19positive isolates belong to the same two most numerous subgroups, respectively. However, the three qnrS1-positive isolates were all clonally unrelated.

Discussion

In this work, we have detected for the first time the presence of qnr genes that confer mild resistance to quinolones among enterobacterial isolates collected in the Northeast region of Italy.

Interestingly, all qnrB-positive K. pneumoniae as well as some E. coli strains bore the qnrB19 allele. Of these, all but one were embedded within a transposon Tn2012 structure associated A c c e p t e d M a n u s c r i p t with an ISEcp1 element and reported identical plasmid electrophoretic profiles. Tn2012 was reported for the first time in an E. coli clinical isolate from Colombia in 2008 [START_REF] Cattoir | ISEcp1-mediated transposition of qnrB-like gene in Escherichia coli[END_REF]. Recently, qnrB19 genes were reported in Salmonella strains from The Netherlands [START_REF] Garcia-Fernandez | Characterization of plasmids harbouring qnrS1, qnrB2 and qnrB19 genes in Salmonella[END_REF] and Italy [START_REF] Dionisi | Characterization of the plasmidborne quinolone resistance gene qnrB19 in Salmonella enterica serovar Typhimurium[END_REF]; all of them were transposon-associated.

In one of our analysed isolates, qnrB19 was found in a completely different context. Genetic environment analysis revealed its association with an integron complex class I bearing an ISCR1 mobile element. This is the first time a qnrB19 gene has been reported in an ISCR1 integron environment, whilst other qnr alleles have been previously described associated with this genetic element (i.e. qnrA1, qnrB2, qnrB6 and qnrB10) [START_REF] Lascols | Type II topoisomerase mutations in clinical isolates of Enterobacter cloacae and other enterobacterial species harbouring the qnrA gene[END_REF][START_REF] Quiroga | Complex class 1 integrons with diverse variable regions, including aac(6')-Ib-cr, and a novel allele, qnrB10, associated with ISCR1 in clinical enterobacterial isolates from Argentina[END_REF][START_REF] Castanheira | First report of plasmid-mediated qnrA1 in a ciprofloxacin-resistant Escherichia coli strain in Latin America[END_REF][START_REF] Ode | Analysis of plasmidmediated multidrug resistance in Escherichia coli and Klebsiella oxytoca isolates from clinical specimens in Japan[END_REF][START_REF] Ma | High prevalence of plasmidmediated quinolone resistance determinants qnr, aac(6')-Ib-cr, and qepA among ceftiofurresistant Enterobacteriaceae isolates from companion and food-producing animals[END_REF]. Very recently, experimental data provided a likely mechanism for ISCR1-mediated qnrB2 gene mobilisation [START_REF] Chen | Mobilization of qnrB2 and ISCR1 in plasmids[END_REF]. ISCR1 can transpose adjacent DNA sequences, mediated by a single copy of the element, in contrast to ISEcp1 where two copies of the element are needed to flank the mobilised gene. In addition, the ISCR1 element plays a role in the expression of nearby genes by providing a promoter [START_REF] Rodriguez-Martinez | Common region CR1 for expression of antibiotic resistance genes[END_REF]. Hence, ISCR1 may be responsible for the mobilisation of virtually any DNA in an extremely efficient manner, and resistance genes found in ISCR1 gene cassettes are potentially highly mobile [START_REF] Toleman | ISCR elements: novel gene-capturing systems of the 21st century?[END_REF]. The fact that only one isolate was found with qnrB19 in this genetic background and that it did not present any further mutation increasing resistance to fluoroquinolones (i.e. topoisomerase II mutations) or to other classes of antibiotics (i.e. ESBL) indicates the rather recent acquisition of the qnrB19 gene in this strain.

The intrinsic properties of its genetic environment predict a high diffusion of this qnrB19 embedded in an ISCR1-bearing mobile element and therefore we expect to find increased qnrB19-mediated resistance to fluoroquinolones in the near future.

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

In contrast, ISEcp1 qnrB19 genes were probably acquired some time ago, since most of them displayed concurrent types of resistance mechanisms such as topoisomerase II mutations, high-level resistance to fluoroquinolones and intensive ESBL production, which are usually stimulated by and then become associated with the qnr gene [START_REF] Strahilevitz | Plasmid-mediated quinolone resistance: a multifaceted threat[END_REF]. It is possible that these ISEcp1 qnrB19 genes were initially acquired from a single source in the initial location (i.e.

Colombia where they were first reported [START_REF] Cattoir | ISEcp1-mediated transposition of qnrB-like gene in Escherichia coli[END_REF]) through strain exchange by travellers and patients between South America and Europe. However, this gene now appears uniformly diffuse in our region since qnrB19-positive samples were collected both from inpatients and outpatients and were randomly distributed among different hospital units.

By contrast, non-qnrB19 genes were found both in K. pneumoniae (qnrA1) and E. coli (qnrS1) isolates. Both their phenotypic and genetic profiles indicated that they were more diversified and were likely acquired from diverse sources.

On the whole, K. pneumoniae species were more likely to present qnr genes than E. coli isolates (68% and 5% of the initial K. pneumoniae and E. coli isolates, respectively). To our knowledge, this is one of the widest spreading of qnr genes in K. pneumoniae isolates. In addition, no similar distribution for the qnrB19 gene has ever been reported. Generally, qnr genes are largely associated with ESBLs: the prevalence of bla genes increased from 33% in all tested isolates to 75% in qnr-positive strains. Nevertheless, contrary to most samples reported in the literature [START_REF] Strahilevitz | Plasmid-mediated quinolone resistance: a multifaceted threat[END_REF][START_REF] Robicsek | The worldwide emergence of plasmid-mediated quinolone resistance[END_REF][START_REF] Cattoir | ISEcp1-mediated transposition of qnrB-like gene in Escherichia coli[END_REF], qnr and bla genes did not co-localise on the same plasmid. The integron is flanked on one side by the 5'-conserved sequence (5'CS) followed by the inverted repeat region 2 (IRR2). The psp operon transcriptional activator (psp O-TA) is truncated and immediately upstream of the qnrB19 gene. Downstream, an integrase 1 (int1) with its putative recombination site is present, followed by the sapA (a peptide transport system permease) gene disrupted by recombination events and a second recombination site (GenBank accession no. GU074393). ciprofloxacin, ofloxacin and moxifloxacin, S ≤ 0.5, R > 1; and levofloxacin, S ≤ 1, R > 2.

A

  c c e p t e d M a n u s c r i p t analysed phenotypically. On the whole, a clear association was found between qnr genes and ESBLs: whilst 33% of the initial 232 clinical isolates tested were ESBL-producers, this value increased to 72% (ESBL phenotypic testing) and 75% (ESBL genotypic analysis) in qnrpositive samples.

  qnr-positive samples were mainly resistant to nalidixic acid and susceptible to other types of antibiotics. Resistance to nalidixic acid and the presence of ESBLs are two phenotypically measured properties that possibly indicate the presence of plasmid-mediated mechanisms of 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

Fig. 1 .

 1 Fig. 1. Plasmid electrophoretic profiles of qnr-positive samples. M, molecular weight marker.

Fig. 2 .

 2 Fig. 2. Genetic environment flanking the qnrB19 gene in isolate #132. The qnrB19 gene is

u s c r i p t 1 2 8 .b

 128 Properties of qnr-positive clinical isolates and transformant and transconjugant strainsTable Susceptibility (no. of isolates) of qnr-positive isolates to quinolone drugs according to Clinical and Laboratory Standards Institute (CLSI) a and European Committee on Antimicrobial Susceptibility Testing (EUCAST) acid; CIP, ciprofloxacin; OFX, ofloxacin; LVF, levofloxacin; MXF, moxifloxacin; R, resistant; I, intermediate; S, susceptible.a Resistance ranges according to CLSI breakpoints (mg/L): nalidixic acid, S ≤ 16, R ≥ 32; ciprofloxacin, S ≤ 1, I = 2, R ≥ 4; and ofloxacin, levofloxacin and moxifloxacin, S ≤ 2, I = 4, R ≥ Resistance ranges according to EUCAST breakpoints (mg/L): nalidixic acid, S ≤ 16, R > 16;

Acknowledgments

The authors thank Prof. E. Cambau (University of Paris XII, France) for kindly donating qnrpositive control strains; Prof. L. Martínez-Martínez (University Hospital Marqués de Valdecilla, Santander, Spain) for the gift of E. coli J53 AzR ; and Prof. L. Poirel (Université Paris-Sud, France) for generously sharing his Kieser protocol.

Funding

This work was supported by Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR) (grant FIRB-Ideas RBID082ATK).

A c c e p t e d M a n u s c r i p t resistance to fluoroquinolones. However, in our experience these two features did not make a predictive phenotype, since six of ten nalidixic acid-susceptible and ESBL-negative samples were qnr-positive. Hence, it would be interesting to analyse next the dissemination of plasmidmediated resistance mechanisms on a larger set of 'susceptible' clinical isolates.

Competing interests

None declared.

Ethical approval

Not required.


Czes Law Szmytkowski

Elżbieta Ptasińska-Denga

Total electron-scattering cross section measurements for tetrahydropyran, (CH 2 ) 5 O, molecules

Keywords:

Absolute total cross section (TCS) for electron scattering from tetrahydropyran [THP: (CH 2 ) 5 O] molecules has been measured from 1 to 400 eV, using a linear electron-transmission technique. The TCS energy function for electron-THP collisions is characterized by the broad enhancement spanned between 3 and 15 eV, which can be attributed to resonant processes. Comparison is also made between the present results for THP and earlier TCS measurements for tetrahydrofuran [THF: (CH 2 ) 4 O] and α-tetrahydrofurfuryl alcohol [THFA: C 5 H 10 O 2 ].

Introduction

The discovery that slow electrons can induce strand breaks in the DNA (Boudaïffa et al 2000) stimulated an increasing interest in experimental and theoretical studies concerning electron interactions with living-cell constituents (Sanche 2005, Winstead andMcKoy 2008, andreferences therein). To overcome difficulties in investigations of electron-induced reactions in vivo state, the studies are focused on the basic DNA components as well as on molecules which may serve as simple models of building blocks for living matter, like furanose-structured molecules: tetrahydrofuran [THF: (CH 2 ) 4 O] and its derivatives. It is believed that the comprehensive and reliable electron-scattering data for such isolated subunits may help to understand and model the electron-assisted processes occurring within the cell in the complex DNA (RNA) compound (Sanche 2009). Tetrahydropyran [THP: (CH 2 ) 5 O], with O atom embedded in the six-membered ring, is a homologue of the THF molecule (see figure 1). The THP ring is the core of pyranose sugars and is a structural base for numerous molecules important in synthesis of biologically active and pharmaceutically effective compounds (Smith et al 2006). The THP moiety has been also identified as a highly advantageous component of liquid crystal mixtures for LCD devices (Kirsch and Maillard 2006).

In contrast to THF, for which extensive studies on the electron impact have been performed, the THP has received much less attention in the electron-scattering research.

For THP there are, till now, available only the core excitation spectra taken at 2.5 keV electron energy (Newbury et al 1986) and recent calculations of elastic and ionization cross sections at intermediate energies (Szmytkowski et al 2009). In the literature, one can find also an experiment concerned the electron diffraction study of the THP molecular structure (Breed et al 1979).

The main goal of the present work is to provide absolute values of total cross sections (TCSs) describing electron-THP scattering over impact energies from low (1 eV) to intermediates (400 eV). The results are then compared with experimental TCS for THF and its derivative α-tetrahydrofurfuryl alcohol [THFA: C 5 H 10 O 2 ].

Experimental

The absolute electron-scattering total cross sections (TCSs) reported in this work have been measured from low to intermediate energies employing the electron-transmission (ET) technique in a linear configuration. In the ET method, the experimental procedure is based on the relation between the total cross section Q(E) and the attenuation of an electron beam of given energy E passing through the target under study. This relation is, to the first approximation, given by the Bouguer-de Beer-Lambert (BBL) attenuation formula,

I(E, n) = I(E, 0)exp[-nLQ(E)],
where I(E, n) and I(E, 0) are the intensities of the electron beam currents, transmitted through the scattering cell, taken in the presence and absence of the target, respectively. The target density number, n, is derived from ideal gas law using the target vapour pressure, measured with a MKS Baratron capacitance gauge. As the temperature of the absolute manometer head, T m = 322 K, is a few degrees higher than that of the target vapour in the scattering cell, T t , the thermal transpiration formula of Knudsen (1910) is applied to convert the pressure reading, p m , into the target pressure, p. Then the density is n

= p/k √ T t T m ,
where k is the Boltzmann constant. L represents the length of the electron pathway within the target approximated with the distance (= 30.5 mm) between entrance and exit cell apertures.

In the reported experiment, the electron beam was energy selected before entering the scattering chamber by an electrostatic 127 • cylindrical condenser; ΔE 0.1 eV (FWHM). The electrons emerging from the reaction volume through the exit aperture were directed into a Faraday cup (FC) and their intensity was measured with a Keithley 617 electrometer. To reduce the number of inelastically scattered electrons which might be accepted by the collector, together with the transmitted electrons, a retarding-field energy selector was applied before the FC detector. The acceptance angle of the detector system, defined by the electron lens apertures, is 0.7 msr. Intensity of the ambient magnetic field along the electron pathway in the electron optics region is kept below 0.1 μT. This ensures that the trajectories of the unscattered electrons are straight lines within the scattering and the detector volumes. The electron energy scale is calibrated against the low-energy resonant oscillatory structure visible in the electron current transmitted when the N 2 is admixtured. The calibration has been carried out several times during the experiment. The uncertainty of the energy scale is estimated to be about 0.1-0.2 eV, mostly due to the shift in the contact potentials observed in the course of the long-lasting experiment, related to the target molecules deposition on the electron optics elements. The spectrometer electron optics is housed in a vacuum chamber with the background pressure lower than 40 μPa.

When the vapour sample is fed into the scattering cell, some amount of the target molecules emerge through the cell orifices into the electron optics region. It was noticed that the presence of target particles in surrounding of the filament changes the electron emission and that the electron current leaving the electron gun is sensitive to changes of the partial target pressure in the electron filament region, what in consequence might affect the measured TCS values. To keep the conditions in the electron optics volume invariable throughout the experiment, irrespective of whether the target is present or absent in the scattering cell, the target vapour was supplied alternately into the collision cell or its surrounding, so the residual target pressure in the electron optics region is below 0.1 mPa and practically constant. The pressure of the target in the reaction volume, when the sample was directed into the cell, was usually about three orders of magnitude higher. In addition, before starting the TCS measurements, the electron optics was passivated in the presence of the THP during some pilot runs, until the filament emission and the electron transmission become stabilized. After such procedure applied, it was found that the TCS values obtained at the same energy were, within the random experimental uncertainties, independent of the applied target pressures (0.08-0.3 Pa) and the electron-beam intensities (0.04-20 pA). Other details of the experimental arrangement and data processing employed have been described elsewhere (e.g. Szmytkowski et al 1997, Szmytkowski andMożejko 2001).

Research-grade THP from Sigma-Aldrich, of a stated purity 99% (the boiling point of 361 K), was used in the experiment. Prior to use, however, the sample was subjected to several liquid-nitrogen freeze-pump-thaw cycles to remove dissolved air and other volatile impurities. The sample-handling system has been maintained at an elevated temperature around 310 K what ensured the sufficient inlet pressure of the target vapour.

The quantities necessary for the TCS derivation are taken directly in the experiment, so the TCSs reported in this work (see table 1 andfigure 2) are in absolute scale. The statistical uncertainty (one standard deviation of the weighted mean value) of the TCS does not exceed 1% over the entire energy range investigated. The inability to discriminate against electrons which are scattered elastically through the small angles (≤ 2 • ) in the forward direction may systematically lower the measured TCS by about 4% at low impact energies. The error related to the effusion of the target particles through orifices of the scattering cell, that leads to inhomogeneity of the target density in the reaction volume and to elongation of the effective path over which the scattering events may take place, is about 2.5%. Other possible TCS systematic error components are estimated to be less than 1% each. The overall systematic uncertainty of our measured absolute TCS, estimated as a sum of possible systematic errors, amounts up to 9-11% below 2 eV, 4-6% within 2-150 eV, and 7-9% at higher energies applied.

Results and discussion

The present work reports the absolute electron-scattering total cross sections (TCSs) for tetrahydropyran [THP: c-(CH 2 ) 5 O] measured in the electron-transmission experiment over the 1-400 eV energy range. The numerical TCS values are listed in table 1, while in figure 2 The distinctive feature of the measured e --THP TCS is its relatively large magnitude across the entire energy range investigated. Between 1 and 50 eV the TCS values lie above 50 × 10 -20 m 2 , and even at the highest energy used (400 eV) the TCS exceeds 16 × 10 -20 m 2 . Such high cross-section values can be related, at least in part, to the large geometrical size of the THP molecule.

With respect to the shape, four regions can be distinguished in the experimental TCS energy dependence for THP. In the lowest (1-3 eV) energy region studied, the TCS first decreases only just from 51 × 10 -20 m 2 at 1 eV to about 50 × 10 -20 m 2 in the vicinity of 2.2 eV where some weak minimum is noticeable; then it slowly increases and shows a subtle structure located near 2.6 eV. The most pronounced feature-a broad asymmetric TCS enhancement-is clearly visible in the energy range between 3 and 15 eV. This enhancement is composed of three structures: the shoulder of a value 59-61 × 10 -20 m 2 spanned between 3.8 and 5 eV; the change of the slope distinguishable around 7 eV; and the main maximum (68 × 10 -20 m 2 ) located close to 8.5 eV. In the third energy range, 15-30 eV, worth noting is the very broad shoulder where the TCS is nearly constant (∼ 55 × 10 -20 m 2 ). Beyond 30 eV the TCS decreases with increasing energy as E -a , where a ∼ 0.5; at 400 eV the TCS falls to 16 × 10 -20 m 2 . As it can be seen in figure 2, above 60 eV, the experimental TCS is satisfactorily reproduced with the sum of elastic and ionization cross sections calculated by Szmytkowski et al (2009); differences do not exceed the experimental uncertainties. Such agreement seems to be a bit surprising, as the calculated cross section (elastic plus ionization) does not include cross sections for the variety of inelastic processes allowed in this energy range. It is also worth to mention that, above 50 eV, the measured TCS is in good agreement with the TCS predictions based on the simple additivity group rule (Szmytkowski et al 2009).

Due to a lack of detailed experimental and theoretical investigations of electron-THP scattering, suppositions concerning origins of the observed low-energy TCS features may be based only upon the data for molecules of similar geometry and/or composition, especially those oxygen-containing. Taking into account results available for numerous polyatomic hydrocarbons one can make an assumption that the 3-15 eV enhancement is to a large extent connected with indirect (resonant) scattering processes occurring in this energy range. The resonant scattering appears when the impinging electron is temporarily attached to target molecule forming a short-lived negative ion state (resonance) which decays with an emission of the extra electron-leaving the parent molecule in the excited state, or dissociates into negative and neutral fragments.

Figure 3 compares the present electron-scattering TCS for THP to that for THF molecule (Możejko et al 2006a). The THF, a five-membered hetero cyclic homologue of THP (cf. figure 1), has been recently intensively investigated, especially in the low energy range. The experimental (Lepage et al 1998, Aflatooni et al 2006, Sulzer et al 2006, Dampc et al 2007a-b, Colyer et al 2007, Allan 2007) and theoretical (Bouchiha et al 2006, Trevisan et al 2006, Tonzani and Greene 2006, Winstead and McKoy 2006) studies of the e --THF scattering have shown that the low-energy structures in the THF curve can be explained through the formation of the shape and/or core-excited resonances. A close similarity in the shape of the TCS curves for THF and THP targets along with similarity in their molecular geometry suggests that the same mechanisms may be responsible for the low-energy TCS features in both curves. However, the resonances in THP seem to be formed at slightly lower energies.

In addition, figure 3 comprises the TCS results for α-tetrahydrofurfuryl alcohol [THFA: C 5 H 10 O 2 ] measured earlier by Możejko et al (2006b). The THFA compound is the derivative of the THF molecule in which one hydrogen atom was substituted with the hydroxy-methyl group, CH 2 OH (see figure 1). It is interesting that, in spite of differences in chemical constitution of the THF, THFA and THP molecules, the general character of their TCS energy functions is similar with respect to the shape. A general likeness of the low-energy TCSs for the THF-THFA-THP family might suggest that the substitution of the hydrogen atom in THF with the CH 2 OH group and/or increasing the C-ring does not influence the resonant scattering too much. Support for such statement comes also from earlier studies for other hydrocarbons. Similarity in location of resonant structures has been noticed by Allan and Andric (1996) and Allan (2007) in the elastic and vibrational excitation cross sections measured for several cyclic hydrocarbons. Also, recent low-energy elastic calculations for THF and for its derivative, 3-hydroxytetrahydrofuran [3-hTHF: C 4 H 8 O 2 ], show the same trend in the integral cross sections for both molecules (Vizcaino et al 2008).

The differences visible in figure 3 concern mainly the magnitude of TCSs. At energies below 20 eV, the TCS distinctly increases across the series THF, THP and THFA. The embedding of one methylene group, CH 2 , into the THF ring to form THP increases the size of the resulting target molecule, what reflects in the enhancement of the TCS magnitude for THP. A similar effect arises while replacing one hydrogen atom in THF with the CH 2 OH group. On the other hand, change of the molecular constitution affects the electric charge distribution in the molecule: the electric dipole moment and for: tetrahydropyran (THP), ( • ), present; tetrahydrofuran (THF), ( ), Możejko et al (2006a); α-tetrahydrofurfuryl alcohol (THFA), ( ), Możejko et al (2006b).

electric dipole polarizability (cf. table 2). In consequence, such substitutions influence an effective potential of the electron-molecule interaction. Some difference across the series is visible also in the relative intensities of the structures located within 4-7 eV and 7-9 eV. This effect might be related to substitution of functional groups. It can be supported by the observation that the presence of the hydroxy (OH) group in THFA increases the electron-dissociative-attachment cross section around 6 eV, with respect to that of THF, by more than one order of magnitude (Aflatooni et al 2006). At higher energies, above 60 eV, the ratio of TCSs reflects the atomic constitution of molecules. At these energies the impinging electron sees individual atomic components (or functional groups) of the target molecule rather than the molecule as the whole (see Szmytkowski et al 2009). [START_REF] Lide | CRC Handbook of Chemistry and Physics[END_REF]; electrical polarizability (α), estimation based on the additivity formula [START_REF] Miller | [END_REF].

Conclusions

The present paper provides the absolute TCS for electron scattering from THP molecules over impact energies from 1 to 400 eV. The most characteristic features of the measured TCS are: (i) relatively large magnitude of the TCS over whole energy range investigated;

(ii) the pronounced, resonant-like TCS enhancement spanned between 3 and 15 eV. Comparison of the TCS curve for THP with respective data for THF and its derivative, THFA, allows us to assume that the TCS enhancement for THP is mainly related to the formation of resonances in this energy range. At intermediate energies, the experimental TCS is in good agreement with previous TCS (sum of elastic and ionization cross sections) calculations and TCS estimations based on the additivity group rule (Szmytkowski et al 2009). α-tetrahydrofurfuryl alcohol (THFA), ( ), Możejko et al (2006b).

  Figure 1. Schematic of the tetrahydropyran [THP: c-(CH 2 ) 5 O] geometry. For comparison, the geometry of tetrahydrofuran [THF: c-(CH 2 ) 4 O] and αtetrahydrofurfuryl alcohol [THFA: C 5 H 10 O 2 ] is shown.

  the energy dependence of our experimental TCS is shown together with earlier TCS (elastic plus ionization) calculations at intermediate energies (Szmytkowski et al 2009). Finally, in figure 3, the experimental results for THP molecule are compared to the TCS curves for two hetero-cyclic furanose-structured molecules taken in our laboratory (Możejko et al 2006a-b): tetrahydrofuran [THF: C 4 H 8 O] and αtetrahydrofurfuryl alcohol [THFA: C 5 H 10 O 2 ].

Figure 2 .

 2 Figure 2. Total cross-section for electron scattering from tetrahydropyran, c-(CH 2 ) 5 O, molecules: (• ), experimental, present, error bars represent overall uncertainties; (--), calculated, sum of elastic and ionization cross sections, Szmytkowski et al (2009).

Figure 3 .

 3 Figure 3. Comparison of total electron-scattering cross sections (TCSs) measured

  Figure captions:

Figure 2 .

 2 Figure 2. Total cross-section for electron scattering from tetrahydropyran, c-(CH 2 ) 5 O, molecules: (• ), experimental, present, error bars represent overall uncertainties; (--), calculated, sum of the elastic and ionization cross sections, Szmytkowski et al (2009).

Figure 3 .

 3 Figure 3. Comparison of total electron-scattering cross sections (TCSs) measured for: tetrahydropyran (THP), ( • ), present; tetrahydrofuran (THF), ( ), Możejko et al (2006a);

Table 1 .

 1 Absolute experimental electron-scattering total cross sections for tetrahydropyran, c-(CH 2 ) 5 O, molecules; in units of 10 -20 m 2 .

	E (eV) TCS E (eV) TCS E (eV) TCS E(eV) TCS E(eV) TCS
	1 50.8	3.7 57.6	8.5 67.8	23.5 55.4	100 38.7
	1.2 50.5	3.8 58.7	9.0 66.8	26 55.3	110 36.7
	1.4 50.3	4.0 59.5	9.5 64.4	28.5 54.8	120 35.3
	1.6 50.4	4.3 59.9	10.0 62.8	30 54.5	140 32.7
	1.8 50.2	4.5 60.2	10.5 61.1	35 53.4	160 30.0
	2.0 50.1	4.7 60.8	11 60.0	40 52.3	180 27.9
	2.2 49.8	5.0 61.1	12 58.2	45 51.0	200 26.5
	2.4 50.2	5.5 61.5	13 56.9	50 49.9	220 25.0
	2.6 50.9	6.0 63.0	15 55.8	60 47.5	250 23.0
	2.8 51.2	6.5 64.7	17 55.4	70 44.4	300 20.1
	3.0 51.6	7.0 66.0	19 55.4	80 42.3	350 17.6
	3.3 53.1	7.5 66.6	21 55.5	90 40.4	400 16.3
	3.5 55.6	8.0 67.4

Table 2 .

 2 Parameters of THF [(CH 2 ) 4 O], THP [(CH 2 ) 5 O] and THFA [C 5 H 10 O 2 ] compounds: permanent electric dipole moment (μ), from

Acknowledgments

The authors gratefully acknowledge the support by the Polish Government (MNSzW) Research Funds for 2009-2010.