Nichola H Hancocks

Colin R Thomas

Stuart M Stocks

Christopher J Hewitt
email: c.j.hewitt@lboro.ac.uk

An investigation into the preservation of microbial cell banks for αamylase production during 5 litre fed-batch Bacillus licheniformis fermentations

Keywords: bacteria, flow cytometry, membrane potential, membrane integrity, cryopreservation, fermentation, Bacillus licheniformis

Fluorescent staining techniques were used for a systematic examination of methods used to cryopreserve microbial cell banks. The aim of cryopreservation here is to ensure subsequent reproducible fermentation performance rather than just post thaw viability. Bacillus licheniformis cell physiology post-thaw is dependent on the cryopreservant (either Tween 80, glycerol or dimethyl sulphoxide) and whilst this had a profound effect on the length of the lag phase, during subsequent 5 l fed-batch fermentations, it had little effect on maximum specific growth rate, final biomass concentration or α-amylase activity. Tween 80 not only protected the cells during freezing but also helped them recover post-thaw resulting in shorter process times.

NOMENCLATURE

F

feed rate (l h -1 ) S substrate concentration in the feed solution (gl -1 ) µ desired specific growth rate (h -1 )

Y xs maximum biomass yield with a limiting substrate (g/g) X 0 initial amount of cells at the start of feeding (g) m maintenance coefficient (gg -1 h -1 ) t time after feeding commences (h)

Introduction

Within the fermentation industry there is a need to understand and optimise current methods for the preservation of microbial cell banks. From the production of yoghurt to the most advanced recombinant therapeutics, the generation and subsequent maintenance of an optimal cell bank is a critical task in the successful commercialisation of any product made by fermentation [START_REF] Gnoth | Process Analytical Technology (PAT): Batch-to-batch reproducibility of fermentation process by robust process operational design and control[END_REF][START_REF] Hornbaek | Use of fluorescence ratio imaging microscopy and flow cytometry for estimation of cell vitality for Bacillus licheniformis[END_REF][START_REF] Hornbaek | The effect of inoculum age and solid versus liquid propagation on inoculum quality of an industrial Bacillus licheniformis strain[END_REF]Webb et al., 1993). It is therefore surprising that relatively little systematic work has been published concerning the choice of preservation methods and their optimisation. Some generally early work [START_REF] Lovelock | Prevention of freezing damage to living cells by dimethyl sulphoxide[END_REF][START_REF] Polge | Revival of spermatozoa after vitrification and dehydration at low temperatures[END_REF][START_REF] Morgan | Preservation of micro-organisms by drying; A review[END_REF] has resulted in the formulation of two routinely used preservation methods, both of which are largely unsubstantiated with respect to final productivity. Both freeze-drying and cryogenic preservation rely on the prevention of the formation of ice crystals [START_REF] Fuller | Cryoprotectants: The essential antifreezes to protect life in the frozen state[END_REF][START_REF] Hubálek | Protectants used in the cryopreservation of microorganisms[END_REF][START_REF] Kirsop | Maintenance of microorganisms: A manual of laboratory methods[END_REF], a process that disrupts the delicate membrane structures within cells and hence compromises their viability. Starting from a liquid suspension of cells or spores, freeze drying involves the sublimation of water at low temperatures and pressures and is generally considered inferior to cryogenic preservation, where cells are suspended in an appropriate medium supplemented with 20% (v/v) glycerol and rapidly frozen at -80°C [START_REF] Polge | Revival of spermatozoa after vitrification and dehydration at low temperatures[END_REF]. A brief literature search shows that there are few variations on these themes. Other cryoprotectants, such as Tween 80 [START_REF] Beal | Resistance to freezing and frozen storage of Streptococcus thermophilus is related to membrane fatty acid composition[END_REF][START_REF] Endo | Trans fatty acids promote the growth of some Lactobacillus strains[END_REF][START_REF] Smittle | Relationship of cellular fatty acid composition to survival of Lactobacillus bulgaricus in liquid nitrogen[END_REF], yeast and malt extract [START_REF] Hubálek | Protectants used in the cryopreservation of microorganisms[END_REF][START_REF] Johannsen | Malt extract as protective medium for lactic acid bacteria in cryopreservation[END_REF] and more defined agents such as DMSO [START_REF] Lovelock | Prevention of freezing damage to living cells by dimethyl sulphoxide[END_REF], have also been studied. However, there is little informed or systematic work to back up their use, while the current industrial practice of testing working cell banks through productivity measurements in small scale process representations of production scale often leads to the discovery of a poor cell bank which must then be discarded. Therefore, it would seem that there is a real need for a systematic examination of the methods used to preserve cell banks to ensure subsequent reproducible fermentation performance.

Measurable effects on 'sensitivity', defined here as a loss of productivity rather than just cell viability, may be subtle so appropriate techniques to measure the effects are required.

Multi-parameter flow cytometry has many advantages over conventional microbiological analyses such as dilution plating (c.f.u. per ml) and these have been extensively reviewed elsewhere [START_REF] Nebe-Von-Caron | Analysis of bacterial function by multi-colour and single cell sorting[END_REF]Hewitt andNebe-von-Caron 2001, 2004). Briefly, using various mixtures of fluorescent dyes, it is possible to resolve an individual microbial cell's physiological state beyond culturabilty, in 'real-time', based on the presence or absence of an intact polarised cytoplasmic membrane and the transport mechanisms across it. The presence of both an intact polarised cytoplasmic membrane and active transport are essential for a fully functional (healthy) cell. The speed and sensitivity of this technique was used here to discover those preservation/recovery combinations resulting in the correct physiological state post thaw, which could correlate to final process productivity. In this way candidate preservation/recovery methods were investigated and tested on a standard process model, namely the use of Bacillus licheniformis to produce the commercially important enzyme α-amylase [START_REF] Hewitt | The production of alpha-amylase by Bacillus amyloliquefaciens B20 in a complex and totally defined synthetic culture medium[END_REF] in 5 litre fed-batch fermentation processes.

Materials and methods

Organism and growth conditions

Cell banks of an industrial production strain (Novozymes A/S, Bagsvaerd, Denmark) of Bacillus licheniformis SJ 4628, an asporulating α-amylase overproducing strain, were maintained in 1 ml vials at an OD 600nm of 1 at -80 o C in yeast/malt extract broth (Oxoid, UK) supplemented with either 20% (v/v) glycerol, 25% (v/v) glycerol, 15% (v/v) DMSO or 20% v/v Tween 80.

Inocula were prepared by mixing 1 ml from the cell bank with 9 ml M9 buffer (g l -1 : KH 2 PO 4 , 3; Na 2 HPO 4 , 6; NaCl, 5) in distilled water. One ml 1 M MgSO 4 was added after sterilisation. The cells were grown without shaking in a 500 ml Erlenmeyer flask on 50 ml yeast malt agar (g l -1 : malt extract, 40; yeast extract, 20; M Lab agar, 20) in distilled water. After 16 h at 37 °C, 20 ml M9 buffer was added aseptically to the surface of the agar and the culture mixed to suspend the cells.

For shake-flask toxicity experiments, cells were grown in 50 ml yeast/malt extract broth in a 500 ml baffled Erlenmeyer shake flasks at 37°C and 200rpm. For all shake flask experiments a 2% (v/v) inoculum (prepared from cryopreserved stocks as above) with an OD 600 of 1 was used.

For laboratory scale, fed-batch experiments cells were grown in yeast/malt extract medium (gl water. The fed batch fermentations were carried out in a 5 l cylindrical glass bioreactor (160 mm diam and 300 mm ht), with a working volume of 4 l. The vessel was fitted with two 82 mm, six flat-bladed paddle-type impellers which were 80 mm apart, with the lowest impeller situated 80 mm from the bottom of the vessel. The vessel was also fitted with four 90° baffles of 15 mm width spaced equidistantly around the vessel. The fermentation vessel was equipped for the measurement of DOT, pH and temperature. DOT was maintained at 90% saturation by varying the impeller speed from 0 to 1000 rpm, pH was maintained at 7.0 by addition of 2 M NaOH and 2 M H 3 PO 4 on demand and temperature was maintained at 37 °C. Foaming was controlled by addition of 2% (v/v) polypropylene glycol 2025 on demand. For all fed batch fermentations a 5% (v/v) inoculum was used with an OD 600 of 1. Fed-batch fermentations were started as batch cultures and an exponential feeding profile was calculated from the following equation (Strandberg et al., 1994),

t o xs e X M Y s F 1
where S= 500g/l, Y x/s = 0.5, m= 0.02 and Xo =5 g and µ = 0.3 h -1 .

Feeding was started when the initial glucose was exhausted as measured by a sudden rise in DOT (spike). When the DOT reached the 20% saturation level the feed rate was held constant for the remainder of the experiment.

Analytical Techniques

Biomass was measured from the OD 600 ; direct measurements of dry cell wt, g l -1 , and plate counts, (c.f.u. ml -1 ) were also made. Glucose concentration was measured by HPLC with an AminexHPX-87P carbohydrate analysis column (Bio-rad, UK) and a refractive index analyser. α-Amylase activity was determined spectrophotometrically using the method of Lorentz, (1998).

Fluorescent measurements were made using a Coulter (High Wycombe, UK) EPICS ELITE flow cytometer with 488 nm excitation from an argon-ion laser at 15 mW.

Samples taken from the culture were immediately diluted (at least 1:2000 v/v) with phosphate buffer (PBS, pH 7.0) and stained with a mixture of propidium iodide and bis-(1,3-dibutylbarbituric acid [(DiBAC 4 (3)] Samples were kept in a sonication bath for 10 s prior to analysis in order to avoid problems associated with cell aggregation.

Stock solutions of each dye were prepared as follows: DiBAC 4 (3), was made up at 10 mg ml -1 in DMSO and PI was made up at 2 mg ml -1 in distilled water. The working concentrations of DiBAC 4 (3) and PI were 0.6 g ml -1 and 3 g ml -1 , respectively in Dulbecco's buffered saline (pH 7.2, DBS). All solutions were passed through a 0.2 m filter, immediately prior to use, to remove particulate contamination.

Additionally, software discriminators were set on both the forward and right angle light scatter signals to further reduce electronic and small particle noise. The optical filters were set up so that PI fluorescence was measured at 630 nm and BOX fluorescence at 525 nm. Where there was spectral overlap the systems software compensation was set up to eliminate any interference

Results and discussion

Shake-flask experiments were carried out using B. licheniformis SJ 4628 inocula derived from different cryopreserved cell banks made in either 20% (v/v) glycerol, 25% (v/v) glycerol, 15% (v/v) DMSO or 20% v/v Tween 80. Cell banks frozen without a cryopreservant were also used as a control. In all cases cell banks had >98% viability (as measured by PI exclusion) prior to freezing. Triplicate, reproducible measurements of OD 600nm were made (Figure 1) and cell physiology was monitored throughout the fermentations using multi-parameter flow cytometry (Figure 2). In order to look at the effect of cryopreservant on α-amylase activity further fed-batch fermentations were carried out in 5 l bioreactors by growing the same cell banks, with and without the 16 h incubation pre-inoculation step on yeast malt extract agar, to an OD 600 nm of 1 prior to inoculation. Duplicate reproducible measurements of OD 600nm , DCW (g l -1 ), c.f.u. ml -1 , glucose concentration (g l -1 ) and α-amylase activity (µKat l -1 ) were made (Figure 3, not all data shown). Cell physiology was monitored throughout the fermentation using multi-parameter flow cytometry (Figure 4).

During the shake flask fermentations (Figure 1), both the lag phase and subsequent cell physiology (Figure 2, not all data shown) were affected by the choice of cryopreservant used. The cell banks prepared with 20% (v/v) Tween 80 had the shortest lag phase (~ 3.5 h) presumably because Tween 80 provides better protection from the damaging effects of freezing than the other cryopreservants. Oleic acid is a component Tween 80 and can enhance the ratio of unsaturated fatty acids in the cytoplasmic membrane of lactic acid bacteria allowing them to resist damage during freezing [START_REF] Beal | Resistance to freezing and frozen storage of Streptococcus thermophilus is related to membrane fatty acid composition[END_REF][START_REF] Endo | Trans fatty acids promote the growth of some Lactobacillus strains[END_REF][START_REF] Smittle | Relationship of cellular fatty acid composition to survival of Lactobacillus bulgaricus in liquid nitrogen[END_REF]. Oleic acid is considered necessary for the formation of the cytoplasmic membrane and by various Lactobacillus spp. strains showed that growth was stimulated in the presence of Tween 80 [START_REF] Endo | Trans fatty acids promote the growth of some Lactobacillus strains[END_REF].

Cell banks prepared with 15% (v/v) DMSO showed the longest lag phase (~ 8.5 h) indicating that DMSO is not as efficient at protecting cells during freezing when compared with the other cryoprotective compounds, or had some alternative deleterious effect. The 'beneficial' cryoprotective effects of DMSO were discovered early in the science of cryopreservation and it has since become a popular cryoprotective agent [START_REF] Lovelock | Prevention of freezing damage to living cells by dimethyl sulphoxide[END_REF]. It is claimed that DMSO protects cells by entering the cytoplasmic membrane and directly interacting with the lipid bilayer.

Indeed, DMSO increaseS membrane fluidity which is consistent with positive cryoprotective effects [START_REF] Gurtovenko | Modulating the structure and properties of cell membranes: The molecular mechanism of action of dimethyl sulphoxide[END_REF]. These benefits would not seem to have been reproduced here. Interestingly, the cell banks prepared with 20 % (v/v) glycerol OR 25% (v/v) glycerol show similar lag phase lengths when compared with cell banks prepared with no additional cryoprotectant ( ~ 4 h).

Glycerol was also an early development in the science of cryopreservation and since then has been used widely to protect cells during freezing [START_REF] Polge | Revival of spermatozoa after vitrification and dehydration at low temperatures[END_REF]. As a small poly-hydroxylated molecule, glycerol can enter cells and hydrogen bond to water molecules [START_REF] Fuller | Cryoprotectants: The essential antifreezes to protect life in the frozen state[END_REF]) thereby preventing ice crystal formation. It is also less toxic to cells than DMSO [START_REF] Simione | Cryopreservation manual[END_REF]. Our results suggest that the addition of glycerol to the yeast/malt extract medium surrounding the cells has little effect. This may be because yeast and malt extract themselves are good cryoprotectants for lactic acid bacteria and are often included in freezing protocols [START_REF] Hubálek | Protectants used in the cryopreservation of microorganisms[END_REF][START_REF] Johannsen | Malt extract as protective medium for lactic acid bacteria in cryopreservation[END_REF]). The medium used for these experiments also contained polypropylene glycol 2025 (as an antifoam) which may have helped to protect the cells as both propylene glycol and ethylene glycol have been shown to work as effective cryopreservants [START_REF] Hubálek | Protectants used in the cryopreservation of microorganisms[END_REF].

Surprisingly, the degree of cell death during the freezing process as measured by PI positive staining did not correlate well with length of lag phase. For example 15% v/v DMSO had the same degree of cell death (~47%) post-thaw, as 2% (v/v) glycerol although it took longer for the culture to recover (Figure 5). Indeed there was a considerable proportion of cells (~40%) having depolarised cytoplasmic membranes (i.e. DiBAC 4 (3) positive) 2 h after incubation in the growth medium. This was not observed in any other case but is consistent with a longer lag phase in the case of 15% (v/v) DMSO. Further, 20% (v/v) Tween 80 had a higher degree of cell death (~ 20% post-thaw) than 25% (v/v) glycerol [~13% DiBAC 4 (3)] with the degree of death in cell banks with no cryopreservant being ~38%. In all cases the viable population outgrew the dead cell population and the cells were all viable when the culture reached an OD 600nm of 1 although the length of time to get there was different in each case. It is clear therefore that there is not a simple correlation between cell viability post thaw and length of lag phase; the freezing effects are more complicated than that.

When each of the shake-flask cultures derived from the different cryopreserved stocks reached an OD 600nm of 1.0 they were used to inoculate 5 l fed batch fermentations to determine if the method of cryopreservation affected fermentation process performance as indicated by yield of α-amylase (Figure 3). In all cases no difference was seen in terms of overall growth characteristics (max DCW ~ 27 g l -1 at 17 h), cell physiological state or concentration of α-amylase (max ~10 6 μKat l -1 at 50 h) when using inocula derived from each of the different cell banks and with or without the overnight incubation step on yeast extract malt extract agar (Figures 4 and5, not all data shown). It is clear therefore that in this case inoculum pre-history had no bearing on final process outcome, despite the use of the same number of propagation steps as exploited at the larger scale. However, this may not always be so. B. licheniformis SJ4628 as an industrial production strain is extremely robust and the AmyE gene which encodes for the α-amylase protein is located on the chromosome of the organism rather than on a plasmid. So plasmid instability, which may be affected by cryopreservation, was not an issue here. Other commercially important systems which use plasmid technology may be more sensitive to cryopreservation technique so the results presented here should be used cautiously.

CONCLUSIONS

It is not unreasonable to conclude that any of the cryopreservation techniques described here could be used for productive industrial fermentations of B. licheniformis SJ4628 and probably other Bacillus spp. too. It is also recognised that there will be little motivation to introduce a new cryopreservation protocol if the existing protocol is adequate, especially since this work has demonstrated that it is unlikely that poor cryopreservation is responsible for poor working cell banks which do not have the same process performance as the key strain. So, provided the inoculum is grown up to the same OD and percentage viability, the subsequent production process ought to be robust. It is therefore more likely that the physiological condition or history of the organisms prior to preservation is responsible for the incidence of poor working cell banks. However, if difficulties are being experienced with a particular cryopreservation protocol then Tween 80 may be worth consideration, as it not only protects the cells during freezing but also helps them to recover post thaw resulting in shorter process times.

Strandberg L, Andersson L, Enfors S (1994) The use of fed-batch cultivation for achieving high cell densities in the production of recombinant protein in Escherichia coli. FEMS Microbiol Rev. 14: 53-56.

Webb C, Kamat SP (1993) Improving fermentation consistency through better inoculum preparation. World J Microbial Biotechnology. 9: 308-312.

Figure 1 .

 1 Figure 1. Growth (as OD 600 ) of Bacillus licheniformis SJ4628 during shake-flask

Figure 2 .

 2 Figure 2. Flow cytometric analysis of Bacillus licheniformis SJ4628 stained with

Figure 3 .

 3 Figure 3. Flow cytometric analysis of Bacillus licheniformis SJ4628 stained with

Figure 4 .

 4 Figure 4. (a) DCW (gl -1 ) and (b) alpha-amylase activity (µKatL -1 ) profiles for Bacillus

Figure 5 .

 5 Figure 5. Flow cytometric analysis of Bacillus licheniformis SJ4628 stained with

  Figure 4.

  -1 : (NH 4 ) 2 HPO 4 , 16; malt extract, 40; yeast extract, 20; glucose, 5; KH 2 PO 4 , 1.5; MgSO 4 .7H 2 O, 0.5; CaCl 2 .2H 2 O, 0.45; MnSO 2 .4H 2 O, 0.04; ZnSO 4 .7H 2 O, 0.02; FeSO 4 .7H 2 O, 0.02; Na 2 MoO 4 .2H 2 O, 0.002; CuSO 4 .5H 2 O, 0.01) in distilled water with 3 ml polypropylene glycol 2025. A feed solution (gl -1 : (NH 4 ) 2 HPO 4 , 16; malt extract, 40; yeast extract, 20; glucose, 500; KH 2 PO 4 , 1.5; MgSO 4 .7H 2 O, 0.5; CaCl 2 .2H 2 O, 0.45; MnSO 2 .4H 2 O, 0.04; ZnSO 4 .7H 2 O, 0.02; FeSO 4 .7H 2 O, 0.02; Na 2 MoO 4 .2H 2 O, 0.002; CuSO 4 .5H 2 O, 0.01) was made in distilled

ACKNOWLEDGMENTS

Fluorochromes were provided by Molecular Probes/Invitrogen (Oregon, USA) and the authors thank William Godfrey (Invitrogen Corporation) for his helpful discussions. The authors would like to acknowledge the financial support of the Biotechnology and Biological Sciences Research Council (UK) and Novozymes A/S (Denmark) for this study.


M Hoenigl

P Fussi

G Feierl

U Wagner-Eibel

E Leitner

L Masoud

G Zarfel

E Marth

A J Grisold

Antimicrobial resistance of Streptococcus pneumoniae in Southeast Austria, 1997-2008 

Keywords:

Antibiotic resistance in Streptococcus pneumoniae has increased worldwide but varies within geographical regions. We conducted a retrospective analysis of resistance in S. pneumoniae over a 12-year period to assess local and temporal trends in antibacterial resistance. From 1997 to 2008, a total of 1814 non-duplicate S. pneumoniae isolates were identified at the Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, Austria. Antibiotic resistance was determined by the Clinical and Laboratory Standards Institute (CLSI) disk diffusion test. For penicillin, the minimum inhibitory concentration was determined by Etest. Susceptibility was defined according to CLSI interpretive criteria. For penicillin, resistance rates were consistently low at 0.2% over the 12-year study period. An increase in resistance was remarkable for erythromycin (3.5% in 1997; 14.7% in 2008), clindamycin (1.8% in 1997; 10.6% in 2008) and tetracycline (1.8% in 2000; 11.0% in 2008). For trimethoprim/sulfamethoxazole, resistance increased slightly to 9.2% in 2008. Quinolones showed a low resistance rate of 0.2% that persisted over the whole study period. In contrast to previously published national data, resistance to penicillin was observed to remain at a remarkably low and constant level.

Although international surveillance programmes have set up sustainable and interlinked data networks, our results suggest that regional surveillance may still be needed as decision support for appropriate empirical antibiotic therapy in the local health setting.

A c c e p t e d M a n u s c r i p t 2 E-mail address: andrea.grisold@medunigraz.at (A.J. Grisold).

 This manuscript was presented in part at the 49th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), 12-15 September 2009, San Francisco, CA.

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

Introduction

Streptococcus pneumoniae continues to be a significant cause of morbidity and mortality in humans [START_REF] Musher | Infections caused by Streptococcus pneumoniae: clinical spectrum, pathogenesis, immunity, and treatment[END_REF]. It is a common causative organism of respiratory tract infections, including acute otitis media, acute bacterial sinusitis and community-acquired pneumonia [START_REF]Bilthoven, The Netherlands: European Antimicrobial Resistance Surveillance System (EARSS)[END_REF][START_REF] Fletcher | Controlling invasive pneumococcal disease: is vaccination of at-risk groups sufficient?[END_REF].

Early initiation of appropriate empirical antibiotic therapy is decisive for the outcome of pneumococcal infection, with antibiotic regimens primarily consisting of penicillin, macrolides and quinolones [START_REF] Sjöström | Clonal and capsular types decide whether pneumococci will act as a primary or opportunistic pathogen[END_REF]. The challenge of choosing an effective empirical antibiotic therapy has recently been complicated by two developments. First, rates of resistance to antibiotics widely used for the therapy of pneumococcal infections have increased significantly in recent years [START_REF]Bilthoven, The Netherlands: European Antimicrobial Resistance Surveillance System (EARSS)[END_REF][START_REF] Metz-Gercek | The European surveillance activities EARSS and ESAC in the context of ABS International[END_REF][START_REF] Appelbaum | Resistance among Streptococcus pneumoniae: implications for drug selection[END_REF]. Second, rates of resistance vary not only between continents but also between European countries, as shown by the European Antimicrobial Resistance Surveillance System (EARSS). In particular, resistance to penicillin and erythromycin has spread rapidly but varies within European countries from 1-39% and 1-25%, respectively [START_REF]Bilthoven, The Netherlands: European Antimicrobial Resistance Surveillance System (EARSS)[END_REF].

The aim of this study was to assess whether regional resistance patterns from Southeast Austria reflect the national patterns reported previously and to what extent resistance varies within national borders. In this study, we present results of antibiotic susceptibility testing for pneumococci over a 12-year period in Southeast Austria and link the results to national and international resistance reports.

A c c e p t e d M a n u s c r i p t 5 were from hospitalised patients.

Patients and methods

Patients and bacterial isolates

Identification and antimicrobial susceptibility testing

Identification of S. pneumoniae was based on susceptibility to optochin, bile solubility, API 20 Strep test (bioMérieux Vitek, Inc., Hazelwood, MO) or the GPC card on the VITEK II instrument (bioMérieux, Marcy l'Etoile, France) [START_REF] Arbique | Accuracy of phenotypic and genotypic testing for identification of Streptococcus pneumoniae and description of Streptococcus pseudopneumoniae sp. nov[END_REF][START_REF] Gardam | Optochin revisited: defining the optimal type of blood agar for presumptive identification of Streptococcus pneumoniae[END_REF]. Susceptibility testing was performed by disk diffusion. Resistance was determined according to relevant testing conditions and the new susceptibility interpretation standards proposed by the Clinical and Laboratory Standards Institute [START_REF]Performance standards for antimicrobial susceptibility testing. Eighteenth informational supplement[END_REF]. Minimum inhibitory concentrations (MICs) for penicillin were determined by the Etest method (AB BIODISK, Solna, Sweden). Macrolide resistance phenotypes were determined by the double-disk method [START_REF] Pihlajamäki | Antimicrobial resistance of invasive pneumococci in Finland 1999-2000[END_REF].

Isolates that were non-susceptible to penicillin and to two or more other antibiotics from erythromycin, tetracycline, trimethoprim/sulfamethoxazole (SXT) and quinolones were pneumoniae isolates were stored at -70 C.

Results

Penicillin resistance

Streptococcus pneumoniae isolates with resistance to penicillin were found only sporadically and did not increase over the study period; 3/1814 isolates (0.2%) showed resistance and 46 (2.5%) showed intermediate susceptibility to penicillin. The distribution of MIC values as well as MIC values for 50% and 90% of the organisms (MIC 50 and MIC 90 , respectively) are shown in Table 2.

Erythromycin resistance

Resistance to erythromycin was found to have increased steadily from 3.5% in 1997 to 14.7% in 2008. The overall resistance rate was 9.3% (169/1814 isolates). Resistance rates to clindamycin increased from 1.8% in 1997, reaching 10.6% in 2008. The M-phenotype was found in 45% (76/169) and the MLS B phenotype in 55% (93/169) of the erythromycinresistant isolates (Table 1). An increase in the MLS B phenotype compared with the Mphenotype was noted over the study period.

Susceptibility to other antibiotics

Tetracycline resistance increased steadily over the study period. Whereas 1.8% of S. pneumoniae isolates showed resistance to tetracycline in 1997, the rate increased to pneumoniae isolates in 2008 (Table 1).

Rates of resistance to quinolones (represented by ciprofloxacin and moxifloxacin) remained very low, with no more than one S. pneumoniae isolate per year being resistant (4/1814; 0.2%) (Table 1).

Multidrug-resistant pneumococci

MDRP

Discussion

The antibiotic susceptibility of S. pneumoniae over a 12-year period was assessed and very low rates of penicillin resistance were found, whereas resistance to other antibiotic classes developed in accordance with national and international trends.

Since the first description of an S. pneumoniae strain with reduced susceptibility to penicillin in 1967, resistance to penicillin among pneumococci has increased and spread worldwide. The steady increase of penicillin-resistant clones in the last two decades has been associated with the extensive use of penicillin for infections caused by pneumococci [START_REF]Bilthoven, The Netherlands: European Antimicrobial Resistance Surveillance System (EARSS)[END_REF].

A c c e p t e d M a n u s c r i p t 8 High rates of penicillin-resistant pneumococcal isolates were reported from the USA (21%), Mexico, Brazil, South Africa, Japan and South Korea [START_REF] Jenkins | Trends in antibacterial resistance among Streptococcus pneumoniae isolated in the USA: update from PROTEKT US years 1-4[END_REF][START_REF] Low | Changing trends in antimicrobial-resistant pneumococci: it's not all bad news[END_REF][START_REF] Shin | Prevalence, characteristics, and molecular epidemiology of macrolide and fluoroquinolone resistance in clinical isolates of Streptococcus pneumoniae at five tertiary-care hospitals in Korea[END_REF]. Within European countries, the frequency of penicillin-resistant pneumococcal isolates varies widely.

Resistance rates of >15% were found in Turkey, Bulgaria and Romania and rates of 5-10% in Spain, Ireland, Croatia and France, whilst resistance rates of <1% were found in most northern European countries, Germany and the Czech Republic [START_REF]Bilthoven, The Netherlands: European Antimicrobial Resistance Surveillance System (EARSS)[END_REF]. In several countries (including Turkey, Hungary, Croatia and Ireland), penicillin resistance has increased dramatically over recent years [START_REF]Bilthoven, The Netherlands: European Antimicrobial Resistance Surveillance System (EARSS)[END_REF][START_REF]Bilthoven, The Netherlands: European Antimicrobial Resistance Surveillance System (EARSS)[END_REF][START_REF]Bilthoven, The Netherlands: European Antimicrobial Resistance Surveillance System (EARSS)[END_REF]. Overall, an increase in penicillin resistance from 5.7% in 2007 to 6.3% in 2008 was reported in invasive isolates in Europe [START_REF]Bilthoven, The Netherlands: European Antimicrobial Resistance Surveillance System (EARSS)[END_REF].

For the northern part of Austria, Buxbaum et al. [START_REF] Buxbaum | Serotype distribution and antimicrobial resistance of Streptococcus pneumoniae in Austria[END_REF] found a slight increase in penicillinresistant pneumococcal isolates from 2% in 1996 to 2.2% in 2002. For invasive S. pneumoniae isolates, an overall 6-year resistance rate of 0.9%, reaching 1.6% in 2007, was reported from Austria [START_REF]Bilthoven, The Netherlands: European Antimicrobial Resistance Surveillance System (EARSS)[END_REF][START_REF] Metz-Gercek | Ten years of antibiotic consumption in ambulatory care: trends in prescribing practice and antibiotic resistance in Austria[END_REF]. However, we found constant 5-to 12-fold lower rates of penicillin-resistant S. pneumoniae isolates in Southeast Austria (0.17% over the 12-year study period and no resistant isolate since 2006). These data may indicate the importance of regional surveillance in addition to international surveillance programmes such as EARSS as decision support for appropriate empirical antibiotic therapy in the local health setting. However, two possible limitations must be considered when comparing these data with previously published Austrian data. First, different inclusion criteria may have influenced divergent results compared with the study of Buxbaum et al. [START_REF] Buxbaum | Serotype distribution and antimicrobial resistance of Streptococcus pneumoniae in Austria[END_REF]. Whilst patients below 18 years of age were excluded in their study, this group of patients accounted for the majority of patients in the current study. Second, However, the low rate of penicillin resistance in our study would then be contrary to results by García-Suárez et al. [START_REF] García-Suárez | Serotype distribution and antimicrobial resistance of invasive and non-invasive pneumococcal isolates in Asturias, Spain[END_REF] and Lambertsen et al. [START_REF] Lambertsen | Noninvasive erythromycin-resistant pneumococcal isolates are more often non-susceptible to more antimicrobial agents than invasive isolates[END_REF] who both showed that resistance rates in non-invasive isolates were higher than resistance rates in invasive S. pneumoniae isolates.

Spread of penicillin-resistant pneumococci has led to more extensive use of macrolides for infections caused by S. pneumoniae [START_REF] Greenberg | The association between antibiotic use in the community and nasopharyngeal carriage of antibiotic-resistant Streptococcus pneumoniae in Bedouin children[END_REF]. However, increasing use of macrolides has been associated with an increase in pneumococcal resistance to these agents, and macrolideresistant S. pneumoniae are now more common than penicillin-resistant S. pneumoniae in many parts of the world [START_REF] Greenberg | The association between antibiotic use in the community and nasopharyngeal carriage of antibiotic-resistant Streptococcus pneumoniae in Bedouin children[END_REF].

High rates of erythromycin-resistant pneumococci were found in Asian countries (e.g. 80% in Japan and Korea), South Africa (52%) and the USA (35%) [START_REF] Jenkins | Trends in antibacterial resistance among Streptococcus pneumoniae isolated in the USA: update from PROTEKT US years 1-4[END_REF][START_REF] Low | Changing trends in antimicrobial-resistant pneumococci: it's not all bad news[END_REF][START_REF] Shin | Prevalence, characteristics, and molecular epidemiology of macrolide and fluoroquinolone resistance in clinical isolates of Streptococcus pneumoniae at five tertiary-care hospitals in Korea[END_REF]. Erythromycin resistance rates between 10% and 25% were reported from most European countries.

Highest resistance rates were reported from France (31%), Hungary (31%), Turkey (27%) and Italy (25%); resistance rates <5% were only observed in the Czech Republic, Bulgaria and Baltic countries [START_REF]Bilthoven, The Netherlands: European Antimicrobial Resistance Surveillance System (EARSS)[END_REF][START_REF]Bilthoven, The Netherlands: European Antimicrobial Resistance Surveillance System (EARSS)[END_REF]. A remarkable increase in resistance to erythromycin over the last years was observed in several European countries (Finland from 6% in 1999 to 24% in 2008, and Turkey from 7% in 2003 to 27% in 2008) [START_REF]Bilthoven, The Netherlands: European Antimicrobial Resistance Surveillance System (EARSS)[END_REF][START_REF]Bilthoven, The Netherlands: European Antimicrobial Resistance Surveillance System (EARSS)[END_REF][START_REF]Bilthoven, The Netherlands: European Antimicrobial Resistance Surveillance System (EARSS)[END_REF]. An overall European resistance rate of 16.4% was reported in invasive isolates in 2008 [START_REF]Bilthoven, The Netherlands: European Antimicrobial Resistance Surveillance System (EARSS)[END_REF].

As published previously, resistance of S. pneumoniae to erythromycin in Austria increased from 3.2% in 1996 to 12.8% in 2007 and 9.8% in 2008 [START_REF]Bilthoven, The Netherlands: European Antimicrobial Resistance Surveillance System (EARSS)[END_REF][START_REF] Buxbaum | Serotype distribution and antimicrobial resistance of Streptococcus pneumoniae in Austria[END_REF][START_REF] Metz-Gercek | Ten years of antibiotic consumption in ambulatory care: trends in prescribing practice and antibiotic resistance in Austria[END_REF]. In our study, rates of A c c e p t e d M a n u s c r i p t 10 erythromycin-resistant S. pneumoniae isolates increased in accordance with national and international trends from 3.5% in 1997 to 18.2% in 2004 and 14.7% in 2008. Over the study period, 9.3% of S. pneumoniae isolates from Southeast Austria were found to be resistant to erythromycin.

Erythromycin-resistant pneumococci often show co-resistance to tetracycline. More than 60% of isolates in the USA and more than 80% in Spain and Italy showed this resistance pattern [START_REF] Doern | Antimicrobial resistance among clinical isolates of Streptococcus pneumoniae in the United States during 1999-2000, including a comparison of resistance rates since 1994-1995[END_REF][START_REF] Marchese | Antibiotic susceptibility patterns and serotypes of antibiotic resistant and/or invasive Streptococcus pneumoniae strains circulating in Italy[END_REF][START_REF] Seral | Distribution of resistance genes tet(M), aph3′-III, cat pC194 and the integrase gene of Tn1545 in clinical Streptococcus pneumoniae harbouring erm(B) and mef(A) genes in Spain[END_REF]. In Europe, resistance rates for tetracycline vary between 1% (Denmark) and 41% (France) [START_REF] Riedel | Antimicrobial use in Europe and antimicrobial resistance in Streptococcus pneumoniae[END_REF]. In Austria, 8.5% of pneumococci were reported to be resistant to tetracycline in 1996 [START_REF] Mittermayer | Antibiotic susceptibility of pneumococci isolated in Austria over a four-year period[END_REF]. We found increasing resistance rates to tetracycline, ranging from 1.8% in 1997 to 11% in 2008.

For SXT, reported resistance ranges from 7% in Denmark to 48% in Poland [START_REF] Riedel | Antimicrobial use in Europe and antimicrobial resistance in Streptococcus pneumoniae[END_REF]. In this study we found varying rates of resistance to SXT, from 7.1% in 1997 to 9.2% in 2008.

During the last decade, a worldwide increase of resistance to quinolones in S. pneumoniae has been reported. Notable resistance rates were observed in Hong Kong (14.3%), South Korea (2.9%) and the USA (1.8%) [START_REF] Canton | Worldwide incidence, molecular epidemiology and mutations implicated in fluoroquinolone-resistant Streptococcus pneumoniae: data from the global PROTEKT surveillance programme[END_REF][START_REF] Garcia-Rey | Antibiotic consumption and generation of resistance in Streptococcus pneumoniae: the paradoxical impact of quinolones in a complex selective landscape[END_REF]. In Canada, the rate of ciprofloxacin resistance in S. pneumoniae increased from <1% in 1997 to 4.2% in 2005 [START_REF] Adam | Molecular characterization of increasing fluoroquinolone resistance in Streptococcus pneumoniae in Canada, 1997 to 2005[END_REF]. In a study by Reinert et al. [START_REF] Reinert | Antimicrobial susceptibility of Streptococcus pneumoniae in eight European countries from 2001 to 2003[END_REF] assessing S. pneumoniae isolates from eight European countries, resistance to quinolones ranged from 0% (Austria) to 1.3% (Italy). For Ireland, resistance rates have reached 15.2%, as reported by Goldsmith et al. [START_REF] Goldsmith | Increased incidence of ciprofloxacin resistance in penicillin-resistant pneumococci in Northern Ireland[END_REF]. Quinolone resistance is observed almost exclusively among isolates from adults [START_REF] Garcia-Rey | Antibiotic consumption and generation of resistance in Streptococcus pneumoniae: the paradoxical impact of quinolones in a complex selective landscape[END_REF][START_REF] Appelbaum | Antimicrobial selection for community-acquired lower respiratory tract infections in the 21st century: a review of gemifloxacin[END_REF]. In our study, where most isolates were derived from children, the percentage of resistance against quinolones remained low (0.2%), with no more than one S. pneumoniae isolate per year being resistant.

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

 6 as multidrug-resistant pneumococci (MDRP). Following identification, all S.

  A c c e p t e d M a n u s c r i p t 7 11.0% in 2008. For SXT, resistance rates increased from 7.1% in 1997 to 9.2% of S.

  detected non-duplicate S. pneumoniae isolates in this study and not only of invasive isolates may have strengthened results differing from EARSS data for Austria.

  From 1997 to 2008, 1814 non-duplicate isolates of S. pneumoniae were identified at the Institute of Hygiene, Microbiology and Environmental Hygiene, Medical University of Graz, Austria. Numbers of S. pneumoniae isolates per year are shown in Table1. There were

	943 male patients (52%) and 871 female patients (48%). Patient age ranged from <1 year
	to >90 years. Of the 1814 S. pneumoniae, 892 (49.2%) were isolated in children under 10
	years of age. Sources of the obtained isolates included the upper respiratory tract (1409;
	77.7%), lower respiratory tract (257, 14.2%) and others (148; 8.2%). Moreover, 1150
	(63.4%) of the S. pneumoniae isolates were obtained from outpatients and 664 (36.6%)

  were found in 36/1814 (2.0%) of S. pneumoniae isolates (0.9% in 1997, 0.7% in 1998, 3.5% in 1999, 1.8% in 2000, 0% in 2001, 1.7% in 2002, 3.3% in 2003, 2.8% in 2004, 0% in 2005, 1% in 2006, 5.7% in 2007 and 2.8% in 2008).

Table 1

 1 Percentage resistance to erythromycin, clindamycin, tetracycline, trimethoprim/sulfamethoxazole (SXT) and quinolones in 1814 Streptococcus pneumoniae isolates collected in 1997-2008 in Southeast Austria

	Year No. of isolates % resistant
		Erythromycin Clindamycin Tetracycline SXT Quinolones
	1997 113	3.5	1.8	1.8	7.1 0.9
	1998 140	2.1	0	0.7	1.4 0.0
	1999 115	3.5	0.9	0.0	2.6 0.0

Acknowledgment

The authors thank Katharina Seeber, MSc, for editorial assistance.

Funding

None.

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

In conclusion, the presented data cover resistance in pneumococci over a 12-year period in Southeast Austria. International surveillance programmes for antibiotic resistance have set up sustainable and interlinked data networks [START_REF] Metz-Gercek | The European surveillance activities EARSS and ESAC in the context of ABS International[END_REF]. However, owing to varying resistance rates even within national borders, as we have shown for penicillin resistance in S. pneumoniae, regional surveillance is still needed as decision support for appropriate empirical antibiotic therapy in the local health setting.

Competing interests

None declared.

Ethical approval

Not required.

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


Mark Wainwright
email: mark_wainwright@hotmail.com

'Safe' photoantimicrobials for skin and soft-tissue infections

Keywords:

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

A c c e p t e d M a n u s c r i p t ABSTRACT In light of increasing bacterial drug resistance, novel agents and/or modes of bactericidal action are required. In particular, agents that can act at multiple sites within the bacterial cell appear to offer less potential for the development of resistance. This effect may be achieved using combinations of established drugs. Similar effects have been reported as a result of photodynamic antimicrobial action alone. The photoactivation requirement suggests the utility of such agents in light-accessible presentations such as skin and softtissue infections. This approach also offers considerable potential in decreasing conventional antibacterial use and allowing the conservation of important life-saving systemic agents.

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

Introduction

Dyes that were shown to demonstrate bacteria in the presence of mammalian cells were the progenitors of modern chemotherapeutic agents. Work by Gram, Koch, Ehrlich and others provided a sound basis for the idea of selective toxicity, and several biological stains were introduced as antimicrobial agents in the early part of the last century, namely the acridines proflavine and acriflavine against bacterial infection and the triphenylmethanes crystal violet and brilliant green against both bacterial and fungal infection [START_REF] Browning | Flavine and brilliant green, powerful antiseptics with low toxicity to the tissues: their use in the treatment of infected wounds[END_REF]. Whilst the use of such agents caused tissue staining, many lives were saved, for example during the First World War where 'flavine therapy' was employed in battlefield injuries [START_REF] Browning | The antiseptic properties of acriflavine and proflavine, and brilliant green[END_REF]. Agents such as the acridine dye acriflavine were also tested as systemic (oral) preparations for gonorrhoea, the highly hydrophilic nature of the dye ensuring rapid elimination [START_REF] Assinder | Acriflavine as a urinary antiseptic[END_REF].

Whilst the use of dyes in antimicrobial chemotherapy was naturally cut short by the emergence of penicillin and the modern antibacterials, the use of colour in tissue differentiation has never significantly decreased. Intraoperative use of stains such as methylene blue normally employs aqueous solutions of the dye at typical concentrations of 1% w/v [START_REF] Creagh | Is there a role for in vivo methylene blue staining in the prediction of bladder tumour recurrence?[END_REF]. This concentration of methylene blue (intravenous) is also indicated for the treatment of methaemoglobinaemia, both uses thus demonstrating the low toxicity associated with the phenothiazine derivative. From an antimicrobial point of view, methylene blue has also been administered in the treatment of juvenile falciparum malaria in a significant trial in Burkina Faso [START_REF] Meissner | Safety of the methylene blue plus chloroquine combination in the treatment of uncomplicated falciparum malaria in young children of Burkina Faso[END_REF], whilst the related Use of dyes in a clinical setting is thus possible, but there remains the perceived problem of colouration where this is not the intended effect.

However, there must be a cost-benefit balance made if other treatments are unsuccessful. The use of gentian violet against MRSA provides a good, if relatively rare, example of dye use, yet pre-operative iodine use remains ubiquitous. Given the continuing rise in microbial drug resistance, it seems logical to consider active dyes as local therapeutics where possible and also to use them in order to conserve more valuable systemic agents.

In this area, an enormous potential impact should be made by exploiting the photodynamic effect associated with a range of dye types, some examples of which are already in clinical use in humans for conventional purposes.

Photodynamic agents, or photosensitisers, use light energy to promote the formation of reactive oxygen species (ROS) such as singlet oxygen, superoxide, hydroxyl radicals etc. in situ (Fig. 1). Such species represent a considerable toxic threat to simple microbial cells.

Photoantimicrobials

As noted above, there are various dyes that pre-date conventional drugs as antimicrobials. Since the first literature report of photoantimicrobial action was published in 1900, this too has a longer history [START_REF] Raab | Uber die wirking fluoreszierender stoffe auf infusorien[END_REF]. However, for the reasons mentioned previously, photoantimicrobial agents, which were also dyes, were Plainly, the level of investment made by the pharmaceutical industry in developing antimicrobial chemotherapeutics is enormous. Consequently, the sites and modes of action of modern agents are understood in exquisite detail; indeed, such is the level of understanding that for several decades drug molecules have been specifically designed to be target-specific. As noted in the introduction, this degree of sophistication, while allowing targeting at the molecular level, also exerts selective pressure and promotes microbial resistance development.

Photoantimicrobial agents are not single-target specific. Production of highly reactive singlet oxygen in situ means that a range of biomolecular targets is available in the immediate environment of the photosensitiser (structural proteins, enzymes, nucleic acids, unsaturated lipids etc.) via the diffusion of ROS. Consequently, activity against conventional drug-susceptible microbes is also observed for strains that are conventional-resistant [START_REF] Wainwright | Photobactericidal activity of phenothiazinium dyes against methicillinresistant strains of Staphylococcus aureus[END_REF][START_REF] Wainwright | Photobactericidal activity of methylene blue derivatives against vancomycin-resistant Enterococcus spp[END_REF]. Multiple sites of therapeutic action can be achieved by employing combinations of conventional drugs, as already mentioned, but again this is based on defined, rather than variable, sites of action. In addition, the practicalities of combining different drugs may be self-defeating (drug-drug interactions, contraindication, formulation etc. and, of course, cost).

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

As an example, the standard photosensitiser methylene blue has been shown to cause photodamage in the Gram-negative organism Escherichia coli at the level of the outer membrane [START_REF] Menezes | Photodynamic action of methylene blue: repair and mutation in Escherichia coli[END_REF], cell wall [START_REF] Ergaieg | A comparative study of the photoinactivation of bacteria by meso-substituted cationic porphyrin, rose Bengal and methylene blue[END_REF], ribosome [START_REF] Singh | Singlet oxygen quenchers and the photodynamic inactivation of E. coli ribosomes by methylene blue[END_REF] and nucleic acid [START_REF] Tuite | New trends in photobiology: photochemical interactions of methylene blue and analogues with DNA and other biological substrates[END_REF]. To achieve such varied targeting with conventional agents would require the combination of, for example, a peptide antibiotic, a -lactam agent, a tetracycline and a fluoroquinolone.

Since there is, by definition, a light requirement for the application of photoantimicrobial agents, there is a concomitant limit in terms of potential sites where the approach may be exploited, but this certainly covers skin and soft-tissue infections (SSTIs). Efficient light sources need no longer be of the expensive laser type; there are many highly suitable (and inexpensive) examples based on light-emitting diodes (LEDs). However, there is currently no mechanism that would allow the systemic use of photoantimicrobials, although where there is a localised SSTI or undesirable colonisation they should prove useful, particularly from the angle of conventional antimicrobial conservation.

Permitted agents

The principal dye/photosensitiser in terms of human use is methylene blue, this compound having been employed in various applications, both indicative and therapeutic, since the late 19th century [START_REF] Wainwright | Methylene blue-a therapeutic dye for all seasons?[END_REF]. Other photosensitisers with everyday application in humans include the triphenylmethane dye gentian (crystal) violet and the food colourings erythrosine and phloxine B. Similarly, indocyanine green has various applications in clinical blood flow measurement A c c e p t e d M a n u s c r i p t [START_REF] Unno | Indocyanine green fluorescence angiography for intraoperative assessment of blood flow: a feasibility study[END_REF] (see Table 1). Any or all of these examples could be utilised with relatively little difficulty from a regulatory point of view, since each is already in use in humans, and very seldom with any control of incident light. There are many newer, improved photosensitisers available as a result of two decades or more of concentrated research [START_REF] Wainwright | Photosensitisers in biomedicine[END_REF], but these will require considerable investment in time and money in order to reach the clinic. Introduction of these improved agents will depend very much on acceptance of the 'standard' photosensitisers listed above.

The related anticancer approach employing photosensitisers is known as photodynamic therapy (PDT). The agents used here are porphyrin-basedhaematoporphyrin derivatives, chlorins or porphyrin precursors [START_REF] Brown | The present and future role of photodynamic therapy in cancer treatment[END_REF]. Whilst these are, happily, being increasingly licensed in specified cancers, their selectivity for microbial cells is limited and, consequently, they are not covered in this discussion.

Among the 'standard photosensitisers', the use of both methylene blue and crystal violet has been recounted above. Both erythrosine and phloxine B are globally accepted food colourings, whilst the former is also the staining constituent in dental plaque disclosing tablets. Conversely, indocyanine green, as well as being employed in blood volume/blood flow measurement, is a near-infrared absorber and has been shown to act photothermally in tissue soldering [START_REF] Kirsch | Laser tissue soldering in urinary tract reconstruction: first human experience[END_REF] (Table 1). Product familiarity via clinicians' first-hand experience of these materials can only help to promote their use as photosensitisers.

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

Photoantimicrobial activities

As indicated in Table 2, both of the cationic (positively-charged) photosensitisers, methylene blue and crystal violet (Fig. 3), are established broad-spectrum antimicrobials, i.e. active against all four major classes and subdivisions of microbial pathogens. The remaining agents in current human use are all associated with an overall anionic charge (Fig. 3) and this is established as a predisposing factor for lack of photobactericidal efficacy against Gram-negative organisms [START_REF] Demidova | Effect of cell-photosensitizer binding and cell density on microbial photoinactivation[END_REF].

It is evident from Table 2 that there is a considerable knowledge gap concerning erythrosine, phloxine B and indocyanine green with regard to active range. However, it has been reported that negatively-charged photosensitisers such as the related cyanine derivative merocyanine 540 are active against some viruses [START_REF] Günther | Structure-activity relationships in the antiviral and antileukemic photoproperties of merocyanine dyes[END_REF]. Similarly, since the two xanthene derivatives erythrosine and phloxine B have been reported to be effective against Grampositive oral pathogens such as Streptococcus mutans [START_REF] Wood | Erythrosine is a potential photosensitizer for the photodynamic therapy of oral plaque biofilms[END_REF], and indocyanine green is reportedly highly active against S. aureus and Streptococcus pyogenes [START_REF] Omar | Lethal photosensitization of woundassociated microbes using indocyanine green and near-infrared light[END_REF], this suggests their use against other Gram-positive organisms.

Erythrosine has also been reported to be toxic towards the yeast Saccharomyces cerevisiae [START_REF] Matula | Genetic toxicity of erythrosine in yeast[END_REF]. Brief data for the compounds regarding both mammalian use/dose, in vitro photoantimicrobial activities and light dose are given in Table 1. Reports of the use of photosensitisers in either human or animal infections are very scarce, although methylene blue has been used successfully (topical, 2% w/v, red light) in the treatment of onychomycosis [START_REF] Tardivo | Methylene blue in photodynamic therapy: from basic mechanisms to clinical applications[END_REF] and in a murine model of oral candidiasis (local, 0.05% w/v, red light) [START_REF] Teichert | Treatment of oral candidiasis with methylene blue-mediated photodynamic therapy in an immunodeficient murine model[END_REF].

Conclusion

Photosensitisers such as methylene blue and crystal violet have been shown to inactivate all of the bacterial species mentioned in the preceding paragraphs. Given the current state of knowledge regarding structure-activity relationships in photosensitisers, it would not be ridiculous to predict highly effective action against significant Gram-positive organisms for the remaining 'human-safe' photosensitisers covered here.

According to recent data, SSTIs constitute one-fifth of hospital-associated infections in England [START_REF]Reducing healthcare-associated infections in hospitals in England[END_REF]. Given that many SSTIs offer relatively facile topical access, the use of photosensitisers here, in combination with superficial illumination, surely offers considerable potential for infection control. Such an approach has advantages both from the point of view of the lack of likely bacterial resistance and also in the resulting decrease in the use of conventional antimicrobial agents, thus allowing their conservation for more complicated or life-threatening systemic disease.

A

  c c e p t e d M a n u s c r i p t dye gentian violet has been used in the treatment of clinical meticillin-resistant Staphylococcus aureus (MRSA) infection in Japan [6].

A

  photoantimicrobials with that of conventional agents is possible from Fig.2.

A

  c c e p t e d M a n u s c r i p t Data from many more trials currently remain subject to industrial confidentiality.

Fig. 1 .Fig. 2 .

 12 Fig. 1. Photoantimicrobial mechanisms.

Fig. 3 .

 3 Fig. 3. Proposed 'first-generation' photoantimicrobials.

Table 1

 1 Human usage/dosage of proposed photosensitisers and in vitro photoantimicrobial activities [as minimum bactericidal concentration

	Edited Table 1
	(MBC)]
	Photosensitiser Human use	Concentration	Reference Organism	MBC	Light	Light	Reference
			(M)			(M)		dose
								(J/cm 2 )
	Methylene blue Methaemoglobinaemia 30 000	[36]	MRSA	20	White 6	[8]
		Bladder tumour stain	30 000	[4]	Escherichia coli	100	White 6	[38]
		Sentinel node tracing	30 000	[37]	Pseudomonas	25	White 6	[38]
					aeruginosa
	Crystal violet	Topical antisepsis	2500	[6]	Streptococcus	250	HeNe	1	[39]
		(MRSA)			sanguinis		laser
					Candida albicans 250	HeNe	1	[40]
							laser
	Indocyanine	Angiography	6500	[15]	Staphylococcus	32	810	411	[22]
	green				aureus		nm
							laser

a Standard fluorescent room lighting.

Funding

None.

Competing interests

None declared.

+, reported activity; -, reported inactivity; N/R, not reported.

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


Alicia Coelho

Juan José González-López

Elisenda Miró

Carles Alonso-Tarrés

María Beatriz Mirelis

Nieves Larrosa

Rosa María Bartolomé

Antonia Andreu

Ferran Navarro

James R Johnson

Guillem Prats

Characterisation of the CTX-M-15-encoding gene in Klebsiella pneumoniae strains from the Barcelona metropolitan area: plasmid diversity and chromosomal integration

Keywords:

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

IncFIIk, the latter two not having been described previously in association with bla CTX-M-15 .

Several of these plasmids were not self-transferable. Furthermore, in all isolates belonging to sequence type ST-1, bla CTX-M-15 was found integrated into the K. pneumoniae chromosome. In all the studied isolates, the mobile element ISEcp1 was found upstream of bla CTX-M-15 , whereas IS26 was found inserted within ISEcp1 in several isolates, in previously unreported positions. In conclusion, these findings indicate that among K. pneumoniae strains isolated in the Barcelona metropolitan area, bla CTX-M-15 is associated with diverse genetic elements, including the IncR and IncFIIk replicons, as reported for the first time here, and the chromosome.

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

Introduction

The spread of antimicrobial-resistant pathogens is a serious clinical and public health problem worldwide. Recombination processes, including homologous recombination and transposition, constitute the major pathways for the capture and expression of resistance genes within mobile genetic elements that thereby intensively contribute to their dissemination. Regarding the high prevalence of bla CTX-M-15 in Enterobacteriaceae, several reports have emphasised the importance of the insertion sequences ISEcp1 and IS26 in its mobilisation from the Kluyvera chromosome [START_REF] Abbassi | Genetic characterisation of CTX-M-15-producing Klebsiella pneumoniae and Escherichia coli strains isolated from stem cell transplant patients in Tunisia[END_REF][START_REF] Lartigue | Diversity of genetic environment of bla CTX-M genes[END_REF]. Furthermore, the rapid and international spread of bla CTX-M-15 has been mainly associated with the global dissemination of Escherichia coli clonal strains (i.e. sequence types ST-131 and ST-405) carrying bla CTX-M-15, mostly within IncFII conjugative plasmids [START_REF] Mh | Intercontinental emergence of Escherichia coli clone O25:H4-ST131 producing CTX-M-15[END_REF]. Detection of CTX-M-15producing Klebsiella pneumoniae is also increasingly documented worldwide and some authors have proposed horizontal transmission of bla CTX-M-15 between E. coli and K. pneumoniae strains by conjugal transfer of IncFII plasmids [START_REF] Diestra | Characterization of plasmids encoding bla ESBL and surrounding genes in Spanish clinical isolates of Escherichia coli and Klebsiella pneumoniae[END_REF].

In a recent study [START_REF] Coelho | Detection of three stable genetic clones of CTX-M-15-producing Klebsiella pneumoniae in the Barcelona metropolitan area, Spain[END_REF] investigating the presence of CTX-M-15-producing K. pneumoniae clonal strains in the Barcelona metropolitan area from 2005 to 2008, we screened for bla CTX-M-15 among 111 non-duplicate extended-spectrum -lactamase (ESBL)-producing K. pneumoniae isolates. We reported 37 CTX-M-15-producing K. pneumoniae isolates that, based on pulsed-field gel electrophoresis (PFGE), were classified in eight clonal clusters, corresponding with seven sequence types, as determined by multilocus sequence typing (MLST) [START_REF] Coelho | Detection of three stable genetic clones of CTX-M-15-producing Klebsiella pneumoniae in the Barcelona metropolitan area, Spain[END_REF]. Pulsotypes VII and VIII, which exhibited 79% similarity, belonged to the same sequence type, i.e. ST-1 [START_REF] Coelho | Detection of three stable genetic clones of CTX-M-15-producing Klebsiella pneumoniae in the Barcelona metropolitan area, Spain[END_REF]. In the present report, we describe the location and genetic organisation of bla CTX-M-15 in these 37 K. pneumoniae isolates.

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

Materials and methods

Bacterial isolates

The 37 studied K. pneumoniae isolates represented all of the CTX-M-15-producing isolates found among 111 ESBL-producing K. pneumoniae collected from three hospitals within metropolitan Barcelona between 2005 and 2008, as previously reported [START_REF] Coelho | Detection of three stable genetic clones of CTX-M-15-producing Klebsiella pneumoniae in the Barcelona metropolitan area, Spain[END_REF].

According to 7-gene MLST, these isolates belonged to sequence types ST-1 (n = 12), ST-14 (n = 1), ST-37 (n = 1), ST-147 (n = 1), ST-321 (n = 1), ST-326 (n = 16) and ST-327 (n = 5) [START_REF] Coelho | Detection of three stable genetic clones of CTX-M-15-producing Klebsiella pneumoniae in the Barcelona metropolitan area, Spain[END_REF].

Characterisation of the genetic environment of bla CTX-M-15

The genetic environment surrounding bla CTX-M-15 was investigated in the 37 K. pneumoniae isolates by polymerase chain reaction (PCR) mapping and direct sequencing of both strands of the amplicons. To detect nucleotide sequences previously reported to be associated with bla CTX-M-15 , specific primers for the insertion sequences IS26 and ISEcp1 (located upstream of bla CTX-M-15 ) and for the open reading frame orf477 (located downstream) were used in combination with bla CTX-M-15 -specific primers [START_REF] Abbassi | Genetic characterisation of CTX-M-15-producing Klebsiella pneumoniae and Escherichia coli strains isolated from stem cell transplant patients in Tunisia[END_REF][START_REF] Lartigue | Diversity of genetic environment of bla CTX-M genes[END_REF][START_REF] Diestra | Characterization of plasmids encoding bla ESBL and surrounding genes in Spanish clinical isolates of Escherichia coli and Klebsiella pneumoniae[END_REF].

Determination of plasmid replicon type

Plasmid replicons were identified using the PCR-based replicon typing method as described previously [START_REF] Carattoli | Identification of plasmids by PCR-based replicon typing[END_REF], including screening for the IncR replicon that has been recently documented in Salmonella strains [START_REF] García-Fernandez | Characterization of plasmids harbouring qnrS1, qnrB2 and qnrB19 genes in Salmonella[END_REF]. In addition, the repA gene associated with the replicon from the pKPN4 plasmid was amplified using primers RepFIIk-F (5'-GGTTAAAAACCCGAATCCGG-3') and RepFIIk-R (5'-CAGTCGCGTGTAGTTGTTCC-3').

All positive results were confirmed by sequencing both strands of the amplicons.

A c c e p t e d

M a n u s c r i p t

Identification and characterisation of the location of bla CTX-M-15

Plasmid number and sizing was performed on all K. pneumoniae isolates as well as all associated transconjugants or transformants by PFGE of total DNA digested with S1nuclease as previously described [START_REF] García | Acquisition and diffusion of bla CTX-M-9 gene by R478-IncHI2 derivative plasmids[END_REF]. Restriction fragments from PFGE gels were then transferred onto positively charged nylon membranes and hybridised with specific probes for bla CTX-M-15, aac(6')-Ib-cr and each previously identified replicon (i.e. FII, FIIk, R, N and L/M). A chromosomal location of bla CTX-M-15 in the K. pneumoniae isolates was also screened for by the same method using PFGE and Southern hybridisations following I-CeuI nuclease digestion as previously described [START_REF] Liu | Genomic mapping with I-Ceu I, an intron-encoded endonuclease specific for genes for ribosomal RNA, in Salmonella spp., Escherichia coli, and other bacteria[END_REF]. Specific probes for the 16S rRNA gene, bla SHV-1 and seven housekeeping genes (infB, gapA, rpoB, mdh, phoE, tonB and pgi) were used for chromosomal detection [START_REF] Diancourt | Multilocus sequence typing of Klebsiella pneumoniae nosocomial isolates[END_REF].

Plasmid mobilisation

Based on the S1-nuclease experiment results, several representative isolates from each clonal cluster were selected for conjugation assays. Conjugation was carried out at 37 C and 30 C using a rifampicin-resistant derivative of E. coli HB101 as recipient, selecting on Luria-Bertani (LB) agar plates supplemented with 100 g/mL rifampicin and 10 g/mL cefotaxime. For strains for which no transconjugant could be obtained, plasmids were extracted by the hot alkaline method [START_REF] Kado | Rapid procedure for detection and isolation of large and small plasmids[END_REF] and were transformed by electroporation into E. coli HB101. Transformants were selected on LB agar plates containing 10 g/mL cefotaxime.

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

Results and discussion

Genetic environment of bla CTX-M-15

Four distinct genetic environments surrounding bla CTX-M-15 were found among the studied isolates. In isolates belonging to ST-1, ST-326, ST-37 and ST-321, an entire insertion sequence ISEcp1 was found 48 bp upstream of bla CTX-M-15 , as already described in several reports [START_REF] Lartigue | Diversity of genetic environment of bla CTX-M genes[END_REF]. In isolates belonging to ST-327, ST-14 and ST-147, IS26 was found inserted at, respectively, 209, 497 and 594 nucleotides from the ISEcp1 terminus (submitted under GenBank accession nos. GQ845084, GQ845085 and GQ845086, respectively). Although the presence of IS26 in the genetic environment surrounding bla CTX-M-15 is well known [START_REF] Lartigue | Diversity of genetic environment of bla CTX-M genes[END_REF][START_REF] Diestra | Characterization of plasmids encoding bla ESBL and surrounding genes in Spanish clinical isolates of Escherichia coli and Klebsiella pneumoniae[END_REF], the particular insertion positions observed here have not been previously described, suggesting a different origin or a new reorganisation from those already described. In all isolates studied, open reading frame orf477 was detected downstream of bla CTX-M-15 , as previously described [START_REF] Abbassi | Genetic characterisation of CTX-M-15-producing Klebsiella pneumoniae and Escherichia coli strains isolated from stem cell transplant patients in Tunisia[END_REF][START_REF] Gonullu | Dissemination of CTX-M-15 -lactamase genes carried on Inc FI and FII plasmids among clinical isolates of Escherichia coli in a university hospital in Istanbul, Turkey[END_REF].

Localisation of bla CTX-M-15

PCR-based replicon typing and sequencing showed that replicons IncR, IncFIIA and IncN were present in ST-1 isolates. IncR is a replicon that was recently documented in a qnrS1carrying plasmid from Salmonella enterica isolates and corresponds to a replicon of the K. pneumoniae plasmid pK245, itself described as deriving from the -replicon of the K. pneumoniae plasmid pGSH500 (96% similarity between the protein sequences of the replicases from pK245 and pGSH500) [START_REF] García-Fernandez | Characterization of plasmids harbouring qnrS1, qnrB2 and qnrB19 genes in Salmonella[END_REF][START_REF] Chen | Complete nucleotide sequence of pK245, a 98-kilobase plasmid conferring quinolone resistance and extended-spectrum--lactamase activity in a clinical Klebsiella pneumoniae isolate[END_REF]. In this study, sequences of the IncR PCR products obtained from the present Klebsiella isolates showed 100% identity with the replicase from pK245. Moreover, sequences of the IncFIIA PCR products obtained from ST-1 isolates correspond to replicase RepA from the replicon IncFIIk, as previously A c c e p t e d M a n u s c r i p t identified in K. pneumoniae plasmid pKPN4 (based on GenBank sequence NC_009650) (Table 1) [START_REF] Carattoli | Resistance plasmid families in Enterobacteriaceae[END_REF].

Specific probe hybridisations of S1-digested DNA from ST-1 isolates demonstrated that isolates kp-57, kp-62 and kp-64 carried bla CTX-M-15 and aac(6')-Ib-cr on plasmids that exhibited replicon IncR (pST1-2,3) (Table 1; Fig. 1). The IncFIIk replicon was identified by the same method in ST-1 isolate 19sp on a 190-kb bla CTX-M-15 -carrying plasmid (pST1-1) (Table 1; Fig. 1). No IncN plasmid was found in association with bla CTX-M-15 (Table 1).

The remaining eight ST-1 isolates did not exhibit any plasmid carrying bla CTX-M-15 .

However, following restriction of their total DNA with the I-CeuI nuclease (i.e. chromosomal restriction), all ST-1 isolates, including the four carrying a plasmidic bla CTX-M-15 , specifically hybridised with the bla CTX-M-15 probe on a very large (>2000 kb) I-CeuI-digested fragment.

Although this fragment yielded only weak hybridisation with the 16S rRNA probe, it hybridised strongly with the gapA (Fig. 2) and bla SHV-1 probes (data not shown), thereby definitively confirming the chromosomal localisation of bla CTX-M-15 in these isolates.

Although bla SHV-1 can be found not only in a chromosomal location but also in plasmids, this gene was used for the hybridisation experiment because within the chromosome of K. pneumoniae (based on GenBank sequence NC_009648), gapA and bla SHV-1 are separated by only 437 kb.

In isolates with the chromosomal bla CTX-M-15 , the aac(6')-Ib-cr gene was found only on plasmids of molecular weight from 35 kb to 60 kb (paST1-1,2,3) (Table 1). These plasmids did not correspond with any of the incompatibility groups tested by replicon typing.

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

Regarding the remaining 25 K. pneumoniae isolates, PCR-based replicon typing and sequencing as well as Southern analysis of S1-digested DNA demonstrated that all 16 ST-326 isolates carried bla CTX-M-15 plus aac(6')-Ib and/or aac(6')-Ib-cr (since these isolates carried both wild-type and bifunctional acetyltransferase-encoding genes [START_REF] Coelho | Detection of three stable genetic clones of CTX-M-15-producing Klebsiella pneumoniae in the Barcelona metropolitan area, Spain[END_REF]), co-localised on IncR plasmids of ca. 75, 85 and 90 kb (pST326-1,2,3) (Table 1). All five ST-327 isolates carried bla CTX-M-15 on IncFII plasmids, which were of ca. 60 kb in four isolates (pST327-1) (Table 1; Fig. 1) and 30 kb in the fifth isolate (pST327-2) (Table 1). In the ST-37, ST-147, and ST-321 isolates, both bla CTX-M-15 and aac(6')-Ib-cr were found on IncFII plasmids that ranged in size from 75 kb to 80 kb (pST37-1, pST147 and pST321) (Table 1; Fig. 1).

Moreover, ST-37 isolate 23.1sp carried a second copy of these genes on a 290-kb plasmid that was positive for both the FII and FIIk replicons (pST37-2) (Table 1). Notably, this isolate contained a third plasmid of ca. 190 kb that was positive for the IncFIIk replicon (not shown in Table 1). These results are seemingly incompatible since in principle two plasmids belonging to the same Inc group cannot occur in the same host cell. Therefore, it is probable that another unknown replicon is present and expressed in at least one of these plasmids. Finally, isolate kp-71, representing ST-14, carried bla CTX-M-15 and aac(6')-Ib and/or aac(6')-Ib-cr [START_REF] Coelho | Detection of three stable genetic clones of CTX-M-15-producing Klebsiella pneumoniae in the Barcelona metropolitan area, Spain[END_REF] on a 340-kb plasmid that was negative for all the incompatibility groups tested (pST14) (Table 1; Fig. 1).

Of note, neither IncR plasmids, previously reported in S. enterica and K. pneumoniae, or IncFIIk plasmids, previously reported only in K. pneumoniae, have been described in association with bla CTX-M-15 . Moreover, none of the K. pneumoniae isolates not belonging to ST-1 exhibited a chromosomal copy of bla CTX-M-15 (Table 1).

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

In this study, the IncR and IncFIIk replicons occurred in the majority of isolates (i.e. IncR in 33 isolates and IncFIIk in 26 isolates) (Table 1), with or without being associated with bla CTX-M-15 or other studied resistance genes. By contrast, the IncFII replicon occurred in only a few isolates (n = 8), always associated with bla CTX-M-15 . Although additional studies are needed to confirm these relationships, the present data would suggest that IncR and IncFIIk plasmids may benefit from good stability in K. pneumoniae, whereas IncFII plasmids perhaps are maintained in this host by means of selective advantage provided by bla CTX-M-15 . This is consistent with the observation that IncFIIk and IncR plasmids appear to be associated with strains with a high capacity to diffuse and to persist in time, whereas IncFII plasmids were found in strains that have disseminated less extensively [START_REF] Coelho | Detection of three stable genetic clones of CTX-M-15-producing Klebsiella pneumoniae in the Barcelona metropolitan area, Spain[END_REF]. Notably, the genetic environment of bla CTX-M-15 in all the IncR and IncFIIk plasmids revealed an entire ISEcp1 upstream of the gene, which may facilitate gene mobilisation, whereas in some IncFII plasmids the IS26 insertion within the tnpA of ISEcp1 may inhibit ISEcp1mediated mobilisation, trapping the gene in the plasmid, as previously described [START_REF] Ensor | Predominance of CTX-M-15 extended spectrum -lactamases in diverse Escherichia coli and Klebsiella pneumoniae from hospital and community patients in Kuwait[END_REF].

Transferability of bla CTX-M-15

To determine whether plasmids carrying bla CTX-M-15 were self-transferable, conjugation experiments were performed with several isolates (Table 1). Transconjugants were obtained from isolates kp-57 (ST-1), 19sp (ST-1), kp-8 (ST-326), kp-71 (ST-14), 23.1sp (ST-37), kp-28 (ST-147) and 25sp (ST-321). In contrast, bla CTX-M-15 -carrying plasmids could not be similarly self-transferred from isolates kp-62 (ST-1), kp-64 (ST-1), 8H (ST-326), kp-36 (ST-327) and kp-73 (ST-327). However, they could be mobilised by transformation. Isolate kp-30 (ST-1), for which only a chromosomal location of bla CTX-M-15 was detected, yielded neither transconjugants nor transformants, as expected.

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

In transconjugants obtained from isolates kp-57, 19sp, kp-71, 23.1sp and 25sp, the plasmids carrying bla CTX-M-15 were of the same size and carried the same replicon as the plasmids from the donor strains. However, in the remaining two transconjugants (i.e. TC kp-8 and TC kp-28 ), the plasmids carrying bla CTX-M-15 were larger than the corresponding donor strain's bla CTX-M-15 -containing plasmids (Table 1; Fig. 1).

In the case of TC kp-8 , a 330-kb plasmid was detected that was positive for bla CTX-M-15 and for both replicons IncR and IncFIIk, whereas in donor strain kp-8 (from ST-326), the bla CTX- M-15 -carrying plasmid had a size of 90 kb and was positive only for IncR. Interestingly, the donor strain carried an additional plasmid of 205 kb, positive for IncFIIk (data not shown).

Therefore, we hypothesise that the plasmid detected in the transconjugant was the result of a co-integration event involving both parental plasmids, which carried IncR (90 kb) and IncFIIk (205 kb), respectively. Furthermore, the deduced FIIk replicon amino acid sequences obtained for all ST-326 isolates (including kp-8), using primers RepFIIk, exhibited 97.6% identity (six amino acid changes) with the reference RepA sequence from pKPN4 as well as 97.6% identity with the replicase associated with the -replicon carried by plasmid pGSH500, which in addition carried the first-described IncR replicon (based on GenBank sequence AJ009980.1) [START_REF] Ensor | Predominance of CTX-M-15 extended spectrum -lactamases in diverse Escherichia coli and Klebsiella pneumoniae from hospital and community patients in Kuwait[END_REF]. This FIIk variant sequence, designated FIIk-, was also obtained from the TC kp-8 E. coli, a result that may confirm the hypothesised co-integration event.

Transformation experiments involving kp-8 yielded a transformant carrying only the 90-kb IncR plasmid (TF kp-8 ) (Table 1; Fig. 1).

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

Regarding TC kp-28 , a similar recombination event may have occurred involving the IncFII bla CTX-M-15 -carrying plasmid (80 kb) and an IncN plasmid (30 kb) also present in the donor strain kp-28 (from ST-147) (not shown). In contrast, with isolate 23.1sp (from ST-37), although two bla CTX-M-15 -carrying plasmids were detected in the donor strain, in all the conjugation assays only the smaller of these, i.e. the 80-kb IncFII plasmid, was transferred to the recipient (Table 1; Fig. 1).

Conclusion

The present findings elucidate a series of genetic elements that likely contribute to the acquisition and maintenance of bla CTX-M-15 in K. pneumoniae, a species of particular clinical significance because of its current association with major hospital outbreaks. In particular, we provide evidence of bla CTX-M-15 chromosomal integration in K. pneumoniae and document the first reported association of bla CTX-M-15 with the two recently described replicons IncR and IncFIIk. These findings reflect differences in the genetic vectors involved in the carriage of bla CTX-M-15 in K. pneumoniae versus those described in E. coli.

These differences may contribute to the diffusion of bla CTX-M-15 within K. pneumoniae. 1.

A

  c c e p t e d M a n u s c r i p t E-mail address: gprats@vhebron.net (G. Prats). A c c e p t e d M a n u s c r i p t ABSTRACT The localisation and genetic organisation of bla CTX-M-15 were studied in 37 CTX-M-15producing Klebsiella pneumoniae isolates collected from 2005 to 2008 within the Barcelona metropolitan area. Polymerase chain reaction (PCR)-based replicon typing and Southern hybridisations were used to identify the bla CTX-M-15 location. The genetic environment was analysed by PCR mapping and sequencing, and transferability of bla CTX- M-15 was evaluated by conjugation and transformation assays. The majority of the 37 isolates carried bla CTX-M-15 in a plasmid location, frequently associated with the aac(6')-Ib-cr gene. Plasmids encoding bla CTX-M-15 carried three distinct replicons, i.e. IncFII, IncR and

Future

  epidemiological surveillance for bla CTX-M-15 in K. pneumoniae should be carried out to test this hypothesis.

Fig. 1 .

 1 Fig. 1. (a) Pulsed-field gel electrophoresis (PFGE) analysis of S1-digested DNA from

Fig. 2 .

 2 Fig. 2. (a) Pulsed-field gel electrophoresis (PFGE) analysis of I-CeuI-digested genomic

M a n u s c r iFigure 1 :

 1 Figure 1: (a) PFGE analysis of S1-digested DNA from transconjugants or transformants obtained from the studied CTX-M-15-producing

Funding

This work was supported by Fondo de Investigación Sanitaria (PI050289), Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III -FEDER (Spanish Network for Research in Infectious Diseases) (REIPIRD06/0008) and the Office of Research and Development, Medical Research Service, Department of Veterans Affairs (JRJ).

e This group included 13 isolates (1H-13H).

f The deduced FIIk amino acid sequences obtained from all isolates belonging to ST-326 showed 97.6% similarity with RepA from pKPN4 and was referred to as FIIk-, as explained in the Section 3.3.

g Conjugation and transformation assays allowed isolation of the 80-kb plasmid; the 290-kb plasmid could not be isolated.

A c c e p t e d M a n u s c r i Abbreviations: MK, phage λ DNA marker.

Edited Figure 2


Friederike Traunmüller

Michael V Schintler

Stephan Spendel

Martin Popovic

Oliver Mauric

Erwin Scharnagl

Christian Joukhadar
email: christian.joukhadar@jp-medical-research.com

Linezolid concentrations in infected soft tissue and bone following repetitive doses in diabetic patients with bacterial foot infections 

Keywords:

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The present study aimed at assessing unbound extracellular concentrations of linezolid in inflamed soft tissue and bone of diabetic patients suffering from severe bacterial foot infections. Linezolid was administered intravenously twice daily at a dosage of 600 mg. At steady-state conditions, the microdialysis technique was utilised to sample serially interstitial space fluid from inflamed subcutaneous adipose tissue and metatarsal bone from 0-8 h post dose in three representative patients.

Mean peak concentrations of free linezolid in plasma, healthy subcutis, inflamed subcutis and cancellous bone were 16.6 ± 3.0, 15.5 ± 2.5, 15.8 ± 2.8 and 15.1 ± 4.1 mg/L, respectively. The degree of tissue penetration as expressed by the ratio of the area under the concentration-time curve of free linezolid from 0-12 h (fAUC 0-12 ) in tissue to the fAUC 0-12 in plasma was 1.32 ± 0.09, 1.12 ± 0.22 and 1.09 ± 0.11 for healthy subcutis, inflamed subcutis and bone, respectively. Based on currently available pharmacokinetic/pharmacodynamic targets, we conclude that linezolid administered at 600 mg twice daily may be considered an effective treatment in diabetic patients suffering from bacterial foot infection complicated by osteomyelitis.

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

Introduction

In patients with diabetes, serious complications such as peripheral neuropathy combined with bacterial foot infections account for a large number of hospital stays and are a major cause of non-traumatic amputations of the lower limb. Appropriate management of these complications often requires intravenous (i.v.) administration of potent antimicrobial agents supported by surgical intervention. However, measures like these require co-ordinated interdisciplinary work between surgeons, clinicians, clinical pharmacologists and microbiologists because of uncertainty regarding resistance rates, rapidly evolving outbreaks of multiresistant bacterial strains following long-term therapy with broad-spectrum antibiotics, and incomplete knowledge of drug tissue penetration. Thus, choosing the right antimicrobial agent is essential in these situations.

Linezolid has demonstrated potent in vitro activity against problematic bacteria such as meticillin-resistant Staphylococcus aureus (MRSA) and glycopeptide-resistant Gram-positive bacteria [START_REF] Jones | Zyvox Annual Appraisal of Potency and Spectrum program: linezolid surveillance program results for 2008[END_REF]. The ability of linezolid to penetrate into the extracellular space fluid of soft tissues is well documented in healthy volunteers and critically ill patients [START_REF] Deghanyar | Penetration of linezolid into soft tissues of healthy volunteers after single and multiple doses[END_REF][START_REF] Buerger | Pharmacokinetics of unbound linezolid in plasma and tissue interstitium of critically ill patients after multiple dosing using microdialysis[END_REF]. However, the rate and extent of penetration of linezolid into bone tissue is controversial in the medical literature [START_REF] Lovering | Penetration of linezolid into bone, fat, muscle and haematoma of patients undergoing routine hip replacement[END_REF][START_REF] Kutscha-Lissberg | Linezolid penetration into bone and joint tissues infected with methicillin-resistant staphylococci[END_REF][START_REF] Rana | Linezolid penetration into osteo-articular tissues[END_REF][START_REF] Stolle | Pharmacokinetics of linezolid in bone tissue investigated by in vivo microdialysis[END_REF].

Thus, in the present study we aimed to determine the ability of linezolid to penetrate into bone and the interstitium of inflamed subcutaneous adipose tissue in a small representative cohort of diabetic patients suffering from severe diabetic foot infection (DFI). Recently, the microdialysis technique, which was used in this study, was also employed to assess unbound concentrations of linezolid in cancellous bone tissue of A c c e p t e d M a n u s c r i p t 5 healthy pigs as well as fosfomycin concentrations in healthy and inflamed tissue of diabetic patients presenting with bacterial foot infections complicated by osteomyelitis [START_REF] Stolle | Pharmacokinetics of linezolid in bone tissue investigated by in vivo microdialysis[END_REF][START_REF] Schintler | High fosfomycin concentrations in bone and peripheral soft tissue in diabetic[END_REF].

Subjects and methods

This study was performed at the Division of Plastic Surgery, Department of Surgery, Landeskrankenhaus Universitätsklinikum Graz (State Hospital University Clinic of Graz, Graz, Austria). Analytical work was performed at the laboratory of J&P Medical Research Ltd. (Vienna, Austria).

Study subjects

Microdialysis and sampling procedures

Microdialysis was performed as described in detail elsewhere [START_REF] Schintler | High fosfomycin concentrations in bone and peripheral soft tissue in diabetic[END_REF]. Venous blood was collected at predefined time points from an indwelling i.v. catheter.

Chemical analysis and determination of plasma protein binding

Linezolid concentrations in plasma and microdialysates were measured by highperformance liquid chromatography (HPLC) with ultraviolet detection as described previously [START_REF] Swoboda | A simple isocratic HPLC assay to determine linezolid concentrations in different biomatrices for in vivo and in vitro studies[END_REF]. The lower limit of quantification was 0.05 mg/L both for plasma and microdialysate.

Individual values of plasma protein binding were determined in duplicate by the ultrafiltration method. In brief, aliquots of plasma samples were ultrafiltered using disposable centrifugal filter devices (Ultrafree-MC, molecular cut-off 5000 Da; Millipore, Bedford, MA) at 7500  g for 30 min. Filtrates were analysed for free linezolid. The bound fraction was calculated by subtracting the free fraction from unity.

Pharmacokinetic analysis

Pharmacokinetic calculations were carried out using the commercially available computer software Kinetica version 3.0 (InnaPhase, Philadelphia, PA). For the concentrations at 12 h, the baseline steady-state concentrations were utilised. Areas under the concentration-time curve (AUC) were calculated by use of the linear trapezoidal rule.

Results

No adverse events related to the study drug or to the microdialysis procedure were observed. Key pharmacokinetic parameters of linezolid in healthy and inflamed subcutaneous adipose tissue and in metatarsal bone are summarised in Table 1.

Pharmacokinetic profiles are depicted in Fig. 1.

The degree of tissue penetration as expressed by the free (f) tissue to plasma ratios of the AUC from 0-12 h (fAUC 0-12 tissue /fAUC 0-12 plasma ) was 1.32 ± 0.09, 1.12 ± 0.22 and 1.09 ± 0.11 for healthy subcutis, inflamed subcutis and bone, respectively.

Plasma protein binding of linezolid in the study subjects ranged from 18% to 26%.

Discussion

MRSA infections associated with unfavourable outcomes are becoming increasingly problematic in diabetic foot clinics [START_REF] Dang | Methicillin-resistant Staphylococcus aureus in the diabetic foot clinic: a worsening problem[END_REF]. As moderate-to-severe DFI is frequently linked to osteomyelitis of adjacent bones, the ability of certain antibiotic agents to penetrate bone tissue needs to be taken into consideration when aiming for successful treatment of DFI. However, the vast majority of presently available pharmacokinetic data regarding bone tissues focuses on tissue homogenates, which provide no information on the concentration-time course of the drug at the relevant site of most infections, i.e. the extracellular space fluid [START_REF] Stolle | Pharmacokinetics of linezolid in bone tissue investigated by in vivo microdialysis[END_REF].

Against this background, we utilised the microdialysis technique to determine the pharmacokinetic profiles of unbound linezolid in inflamed subcutaneous adipose A c c e p t e d M a n u s c r i p t 8 tissue and metatarsal bone. The key finding was that free linezolid in plasma equilibrates completely with soft tissues and bone within ca. 1 h after the start of infusion (Fig. 1). Tissue:plasma ratios of the fAUC 0-12 were ca. 1, indicating rapid and complete penetration of linezolid into the interstitial space fluid of investigated tissues. It is noteworthy to mention that for measurements in bone, the microdialysis probe was implanted into vital, macroscopically unaffected bone tissue located in close proximity to surgically resected sequestrating bone structures.

In addition, it was shown that inflammation did not affect interstitial concentrations of linezolid in soft tissues (Table 1; Fig. 1). This is in line with another study investigating concentrations of linezolid in perinecrotic wound tissue obtained by biopsy from patients with DFIs. In this study, mean tissue penetration of linezolid was 102% as determined 3 h after an oral dose of 600 mg [START_REF] Majcher-Peszynska | Pharmacokinetics and penetration of linezolid into inflamed soft tissue in diabetic foot infections[END_REF]. On the other hand, in a study by Stein et al. [START_REF] Stein | Linezolid tissue penetration and serum activity against methicillin-resistant Staphylococcus aureus with reduced vancomycin susceptibility in diabetic patients with foot infections[END_REF], penetration of linezolid into soft tissues was only 18-78% (mean 51%) in diabetic patients. It may be speculated that a reduction in blood flow owing to peripheral vascular disease exerted a major impact on the plasma-to-tissue drug equilibration process [START_REF] Joukhadar | Angioplasty increases target site concentrations of ciprofloxacin in patients with peripheral arterial occlusive disease[END_REF][START_REF] Steiner | Effect of norepinephrine on cefpirome tissue concentrations in healthy subjects[END_REF]. Thus, in the present investigation, healthy subcutaneous adipose tissue of a more proximal region of the ipsilateral lower limb was used as reference tissue in order to eliminate potential bias from altered blood flow. We did not find any clinically relevant difference between concentrations of linezolid in healthy reference tissue and inflamed subcutaneous adipose tissue or bone (Table 1; Fig. 1).

The ratio of the fAUC 0-24 in plasma to the minimum inhibitory concentration (MIC) of the infecting pathogen (fAUC 0-24 plasma /MIC) was shown to be highly predictive of antimicrobial killing and clinical success. In seriously ill patients, the target value of A c c e p t e d M a n u s c r i p t 9 fAUC 0-24 plasma /MIC to achieve clinical cure was determined to be 80-120 [START_REF] Rayner | Clinical pharmacodynamics of linezolid in seriously ill patients treated in a compassionate use programme[END_REF]. However, predictive pharmacokinetic/pharmacodynamic (PK/PD) targets for tissues

are not yet established. In this study, the fAUC 0-24 /MIC target of ca. 100 was attained for relevant pathogens with MICs < 4 mg/L in plasma and all tissues investigated [START_REF] Jones | Zyvox Annual Appraisal of Potency and Spectrum program: linezolid surveillance program results for 2008[END_REF].

In summary, based on PK/PD considerations, we conclude that free concentrations linezolid are sufficient to cover soft tissue and bone infections with MRSA or other Gram-positive bacteria commonly isolated in diabetic patients with foot infections complicated by osteomyelitis. Edited Table 1

  Three male patients with type II diabetes [Caucasians aged 60-67 years, body mass index (BMI) 26.3-37.2 kg/m 2 ] presenting with deep-seated bacterial foot infections were included in the study after oral and written informed consent was obtained. The patients required surgical debridement with partial metatarsal bone resection and adjuvant systemic antimicrobial therapy. Exclusion criteria were known allergy to linezolid, renal dysfunction indicated by a creatinine clearance of <40 mL/min as estimated by the Cockcroft-Gault formula, and a history of neutropenia or thrombocytopenia. All patients had received non-invasive conservative treatment for DFI prior to enrolment into the study. During the conduct of the study, coadministration of antimicrobial agents or medications other than the study drug was permitted if medically indicated. All patients received at least six standard i.v. doses of linezolid (Zyvoxid™, 600 mg/300 mL solution for infusion; Pfizer Corp., Vienna, Austria) over ca. 30 min twice daily prior to microdialysis.

Fig. 1 .

 1 Fig. 1. Pharmacokinetic profiles of linezolid in plasma, soft tissues and metatarsal

C

  max , peak concentration; T max , time to C max ; T 1/2 , half-life at -phase; V ss , volume of distribution at steady-state; AUC 0-x , area under the concentration-time curve from 0x h; f, free; MIC, minimum inhibitory concentration.a Calculated for twice daily administration (AUC 0-12  2).b Example for meticillin-resistant Staphylococcus aureus (MRSA) (MIC = 2 mg/L).

Table 1

 1 Main pharmacokinetic indices of linezolid in plasma and target tissues of diabetic foot infections following repetitive intravenous doses of 600 mg [median (range); n= 3]

	Tissue	C max	T max (h) T 1/2 (h) V ss (L)	AUC 0-24	fAUC 0-
		(mg/L)				(mg h/L) a	24 /MIC b
	Plasma	22.4	0.5	9.3	36.9	229.4	-
	(total)	(15.2-		(7.2-	(30.6-	(198.6-
		26.6)		11.1)	47.5)	331.7)
	Plasma	17.8	0.5	9.3	-	169.1	84.6 (81.4-
	(free)	(12.5-		(7.2-		(162.7-	131.6)
		19.6)		11.1)		263.2)
	Subcutis	13.9	2.5	8.8	-	245.3	122.6
	(healthy)	(13.6-	(2.0-	(7.8-		(202.3-	(101.1-
		19.0)	3.0)	10.9)		349.8)	174.9)
	Subcutis	17.4
	(inflamed)

Funding

The study was supported by an unrestricted grant from Pfizer Corp. (Vienna, Austria).

Competing interests

J&P Medical Research Ltd. is an independent international research institute basically operating according to the Public-Private Partnership concept. CJ is managing director of J&P Medical Research Ltd. and owns 100% options. CJ is also a consultant for pharmaceutical companies. All other authors declare no competing interests.

Ethical approval

The present study protocol was approved by the Ethics Committee of the Medical University of Graz (Austria). Edited Figure 1