D T Likas

N G Tsiropoulos

Fate of three insect growth regulators (IGR) insecticides (flufenoxuron, lufenuron and tebufenozide) in grapes following field application and through the wine making process

Keywords: Chromatographic analysis, Chromatography -HPLC IGR insecticides, pesticides, residues, grapes, wine, vinification

Fate of three Insect Growth Regulators (IGR)

Insecticides (Flufenoxuron, Lufenuron and Tebufenozide) in grapes following field application and through the wine making process Dimitrios T Likas, Nikolaos Tsiropoulos

Introduction

The use of insecticides in viticulture is essential for grape protection against pest diseases, and their use is therefore important on grape productivity and on wine quality.

The grape moth (Lobesia botrana) is a wide-spread and harmful pest of grapes.

During one season in Greece, this insect has three or four generations, if environmental conditions are favourable. This fact leads to the need for pesticide treatments as near as possible to harvest. The use of these products, particularly when the dose and /or the established pre-harvest time is not observed , can lead to hazardous residues (Fernandez et al. 2005a). It is thus of particular interest to assess the presence of residues of these compounds in grapes and their processed products, such as wine. Among the different chemical classes of new insecticides which have been available the last years to control insects on vine, the benzoylureas (flufenoxuron, lufenuron and other active substances) and the diacylhydrazine tebufenozide are the most promising [START_REF] Tomlin | The Pesticide Manual[END_REF]. These insecticides belong to the family of growth control, so they have the lowest risks for the non-target organisms and their use helps in respecting the environmental balances. Put on the market in the 1990's to replace the toxic insecticides, they were included in plans for integrated protection.

Since the amount of residues is greatly affected by the pre-harvest interval, high residues could be present in grapes at harvest time, especially when the active substance applied shows high persistence. Consequently, pesticides residues could also be present in wine if the effect of the wine-making technique on residue reduction is poor (Cus et al. 2010, Cabras and[START_REF] Cabras | Pesticide residues in grapes and wine in Italy[END_REF]. Although data for fungicide residues on grapes and their fate during wine-making are widely available (Garcia-Cazorla and Xirau-Vayreda 1994[START_REF] Cabras | Fate of some new fungicides (cyprodinil, fludioxonil, pyrimethanil and tetraconazole) from vine to wine[END_REF][START_REF] Cabras | Gas chromatographic determination of azoxystrobin, fluazinam , kresoxim-methyl, mepanipyrim and tetrraconazole in grapes must, and wine[END_REF][START_REF] Garcia | Multiresidue determination of fungicides in wine[END_REF][START_REF] Cabras | Pesticide residues in grapes and wine in Italy[END_REF], Cabras et al. 2001, Fernandez et al. 2005a, Mirlean et al. 2005, Vaquero-Fernandez et [START_REF] Garau | Residue-free wines: Fate of some Quinone outside Inhibitor (QoI) Fungicides in the Winemaking process[END_REF], data for insecticide residues are quite limited [START_REF] Cabras | Fate of some insecticides from vine to wine[END_REF][START_REF] Sala | Fate of some common pesticide during vinification process[END_REF][START_REF] Goodwin | Relationship between azinphos-methyl usage and residues on grapes and in wine in Australia[END_REF], Navaro et al. 1999and 2001) and even less as far as benzoulurea and other insect growth regulator insecticides are concerned (Tsiropoulos et al. 1999). However, the type of wine-making process and the different oenological steps (with or without maceration, clarification, filtration) carried out can influence the reduction of pesticide residues (Fernandez et al. 2005aand b, Oliva et al. 2007[START_REF] Cabras | Pesticide residues in grapes, wine and processing products[END_REF][START_REF] Ruediger | Removal of pesticides from red and white wine by the use of fining and filter agents[END_REF]).

This paper aims to contribute to the knowledge of the fate of three common insecticides (flufenoxuron, lufenuron and tebufenozide) residue levels in grapes of two vine cultivars (Roditis and Cabernet Sauvignon) after field applications and during the winemaking process, for which there are very few data concerning the persistence of these active ingredients in grapes and their behaviour during vinification. Furthermore, the influence of different clarifying substances on the removal of residues from wine was studied.

Materials and methods

Chemicals and standards solutions

Analytical standards of flufenoxuron (purity 99.3%) and lufenuron (99.7%) were obtained from Cyanamide (Princeton NJ, USA) and Novartis (Basel, Switzerland), respectively, while tebufenozide (99.9%) was purchased from Dr Ehrenstorfer GmbH.

Individual analytical standard stock solutions 1000 mg L -1 for all insecticides were prepared in acetone and stored at - Cyclohexane and dichloromethane were pesticide residue grade, water and tetrahydrofurane were HPLC-grade and acetonitrile was HPLC-far UV grade. All solvents were purchased from Labscan (Dublin, Ireland). Silica SPE cartridges (500 mg/3 mL) used for the extracts clean-up were purchased from Isolute (IST Ltd.

International Sorbent Technology, Mid Glamorgan, UK). Bentonite, charcoal, polyvinylpolypyrrolidone (PVPP), cellulose, potassium caseinate, gelatine and colloidal silicon dioxide were commercial grade products.

Insecticides formulations Cascade 10 DC (10% flufenoxuron w/v), Match 5 EC (5% lufenuron w/v) and Mimik 24 SC (24% tebufenozide w/v) were purchased from commercial market.

Field experiment

The experimental trials were carried out in two vineyards, located at Nea Aghialos, Magnesia, central Greece. The first vineyard was planted with the white grape cv.

Roditis and the other with the red grape cv. Cabernet Sauvignon. The vines in both vineyards were spaced 1.20 m on the row and 2.70 m between the rows, and, during the experimental periods, they received routine horticultural practices.

There were 3 trials in each vineyard for studying the insecticides' dissipation on grapes. In each trial an insecticide formulation was sprayed on 27 August 2004 at the recommended by the manufacturers rates (of 66 mL hL -1 water for Cascade 10 DC corresponding to 0.066 kg ai ha -1 of flufenoxuron, 100 mL hL -1 water for Match 5 EC corresponding to 0.05 kg ai ha -1 of lufenuron and 60 mL hL -1 water for Mimik 24 SC corresponding to 0.144 kg ai ha -1 of tebufenozide), using an automated high pressure mechanically driven applicator (Euro spray Ecology 2000 type 1100 L). Each trial was divided into 4 randomized plots of 15 vines each. Three of them, used as replicates, were treated with the insecticide and the fourth was left untreated to be used as control.

Meteorological data were collected by an agrometeorological station, located near the vineyard. During the experimental period (42 days) the average daily air temperature was 20.6 o C, average relative humidity was 64.2% and total rainfall was 18.8 mm.

Field dissipation study of insecticides in grapes was performed during the 2004 period. Grape samples were collected at 0 (3h after application, when the spraying mixture had dried), 2, 4, 7, 14, 21, 28, 35 and 42 days after application (DAA). The samples consisted of randomly collected parts of at least 12 bunches of separate vines and the overall sample weight was 1,5-2 kg with 1 kg the minimum weight recommended in the FAO/WHO guidelines(FAO/ [START_REF] Fao | Recommended method of sampling for the determination of pesticide residues[END_REF]. Grape samples for residues determination were forwarded to the laboratory, blended after removal of the stems, subdivided into 50 g aliquots as analytical replicates and stored in individual bags at -18 °C until extraction.

Application of the insecticides' formulations in the above vineyard was repeated on 2006 with the same spraying conditions and doses.

Vinification Process.

Determination of insecticides was performed on grapes before vinification, in crude must, clear must, racked and filtered wine and clarified wine.

For studying the fate of insecticides residues from vine to wine, samples of grape were processed to produce must and wine. Wine was also produced from grapes collected from the control plot to be used as control sample. Vinification experiments and the vinification was performed at laboratory scale. The collected grape samples (~50 kg) were divided into two equal parts, pressed and stemmed. The one part was allowed to ferment with the skins (vinification with maceration) while the other was separated from the skin and the resulting must was allowed to ferment (vinification without maceration). One hundred mg of sodium metabisulfite and 200 mg of dry yeast were added per L of must in each part. After the completion of alcoholic fermentation (20-25 days) at room temperature, the obtained wine was racked and filtered. A 2x50 g aliquot of the above cloudy must was taken and centrifuged at 4000 rpm for 5 min in order to quantify residues in the clear must.

Clarification tests were carried out on 500 mL of the filtered wine samples. The clarifying agents and the doses employed (usually applied in oenological practice) in separate triplicate samples were as follows: bentonite at 0.4 g L -1 , charcoal at 0.2 g L -1 , polyvinylpolypyrrolidone (PVPP) at 0.4 g L -1 , colloidal silicon dioxide 0.5 g L -1 plus gelatine at 0.025/0.05 g L -1 or potassium caseinate at 0.4 g L -1 . After clarification, clear wine and unclarified wine samples were analyzed for insecticide residues.

The samples of grapes before vinification, prepared as described for grape samples in the precedent section, must and clear must, unclarified and clarified wine of sprayed grapes, as well as control wines were conserved at -18 °C until analysis.

Sample extraction and clean-up procedure

The investigated insecticides were extracted from the matrices (grapes, must and wine) by a simple and one step extraction using a mixture of cyclohexanedichloromethane (9/1, v/v) as the extraction solvent described by [START_REF] Likas | Residues screening in apple, grape and wine food samples for seven new pesticides using HPLC with UV detection. An application to trifloxystrobin dissipation in grape and wine[END_REF] and presented briefly below. An aliquot (10 g of previously homogenized grape sample or 5 mL of wine) was mixed in a centrifuge tube with the extraction solvent mixture (10 mL for grapes or 5 mL for wine). The tubes were well agitated for 30 min in orbital shaker and centrifuged. After centrifugation, an appropriate volume of organic layer (5 mL and 3 mL for grape and wine samples, respectively) was transferred to a pear shape flask, carefully evaporated to dryness with a rotary evaporator at 40 o C and the residue was quantitatively transferred with about 1 mL cyclohexane to preconditioned with 10 mL cyclohexane Silica SPE cartridges. After loading of the sample extract, the cartridge was rinsed with 10 mL cyclohexane followed by 3 mL of a cyclohexane-tetrahydrofuran (90/10, v/v) solution. The pesticides were eluted with 2 mL tetrahydrofuran and the eluent was dried under a gentle stream of nitrogen. Residues were re-dissolved in acetonitrile-water (50/50, v/v) solution (in 1 mL for grape and in 0.5 mL for wine) and the resulting solutions were filtered prior to injection into the HPLC system. Unprocessed must and centrifugal must samples were extracted as grape or wine samples, respectively.

Instrumentation and Chromatographic Conditions

Chromatographic analysis for the determination of flufenoxuron, lufenuron and tebufenozide was performed with an HP 1100 liquid chromatograph (Hewlett-Packard GmbH, Waldbronn, Germany) equipped with a ternary-delivery system, a variablewavelength UV detector and an HP ChemStation LC 3D chromatography manager data acquisition and processing system with the ability to obtain UV spectra at selected retention time of chromatograms. The analytical column was a Thermo Hypersil HS C 18 column (250 x 2.1 mm I.D with 5 µm particle size) with a guard column and was maintained at 30 o C. The mobile phase was acetonitrile-water delivered at a flow of 0.26 mL min -1 with a gradient composition; from acetonitrile-water (55/45, v/v), held for 5 min, to acetonitrile-water (80/20, v/v) in 10 min, held for 10 min, and finally a

Results and discussion

Analysis

The screening method used for pesticides residues determination is simple and suitable for routine analysis. The cyclohexane-dichloromethane solution at 9/1 ratio effectively extracted lufenuron as well as flufenoxuron and tebufenozide. Accuracy data were provided for flufenoxuron, lufenuron and tebufenozide by recovery experiments (n=5) with grape and wine samples at three different levels (0.05-1.00 mg Kg -1 and 0.02-0.25 mg L -1 ). The mean recovery percentages for grapes analysis ranged from 88 to 103% for flufenoxuron, 85 to 104% for tebufenozide and 92 to 107% for lufenuron with relative standard deviations (RSDs) <10%. The mean recovery values for wine analysis ranged from 91 to 105% for flufenoxuron, 87 to 99% for tebufenozide and 85 to 106% for lufenuron with relative standard deviations (RSDs) <12%. The limits of quantitation of the method (LOQs), calculated as a signal to noise from untreated grape and wine samples equal to 10. The LOQs for grape analysis was 0.02 mg Kg -1 for flufenoxuron and tebufenozide and 0.05 for lufenuron and for wine analysis 0.005 mg L -1 for flufenoxuron and 0.01 mg L -1 for lufenuron and tebufenozide, depending on the final concentration factor for each extract and the sensitivity of each

Field sample analysis and wine-making

Dissipation of flufenoxuron, lufenuron and tebufenozide residues in "Roditis" and "Cabernet Sauvignon" grape samples from the field experiments is presented in Figures2, 3 and 4. It should be noticed that the insecticides were applied when grapes had attained their final size and any diluting effect was negligible thereafter. In addition, no residues of the studied insecticides were detected in any of the analysed control grape or wine samples.

The data relating to the residues in grapes, must and wine for the vinification experiments carried out are reported in Tables 1, 2, 3 and4. Flufenoxuron. After treatment in 2004, flufenoxuron mean concentrations on grapes ranged from 0.33±0.04 to 0.39±0.06 mg kg -1 for "Cabernet" and "Roditis" grapes, respectively. There after residues decreased very slowly with time (Figure 2) showing pseudo-first-order kinetics (R 2 > 0.944) with a dissipation rate of 0.011 d -1 , and at 42 DAA ~60% of the initial deposited concentration remained in grapes. Similar behaviour with very slow decay rate was also observed for teflubenzuron, an other benzoylurea insecticide, on grapes of Roditis variety (Tsiropoulos et al. 1999). A different fate of flufenoxuron residues was observed in dates where a reduction of 96% of initial flufenoxuron residues was found after 60 days [START_REF] Kamel | Degradation of the acaricides abamectin, flufenoxuron and amitraz on Saudi Arabian dates[END_REF] At 35 DAA, which is the recommended by the manufacturer PHI, flufenoxuron residues on grapes were 0.24±0.03 and 0.26±0.03 mg kg -1 for "Cabernet" and "Roditis", respectively. For the 2006 experiment, grapes harvested 40 DAA contained 31August 2008) MRL value (2.0 mg kg -1 ) for flufenoxuron on grapes. Considering our results, we believe that, even after an additional flufenoxuron application 20-30 days before our spraying (i.e. end of July or early August), residues in "Roditis" and "Cabernet" grapes are unlikely to exceed the MRL value even the first day after the second spraying.

Flufenoxuron residues' partitioning during wine-making process was studied after vinification of grapes collected at 21 and 30 DAA for 2004 (Tables 1 and2) and at 21 and 40 DAA for 2006 (Tables 3 and4). The concentration of residues in the must (0.11-0.18 mg kg -1 ) were significantly lower than in grapes (0.23-0.34 mg kg -1 ), indicating that flufenoxuron was distributed in the solid parts of grapes (skin) as well as in the liquid part. The mean values of transfer factor of flufenoxuron from grapes to must resulted from all vinification experiments were 0.48 and 0.52, for "Roditis" and "Cabernet" grapes respectively. Centrifugation of the must resulted in major removal of flufenoxuron residues as it was adsorbed by the suspended solids contained in the must and residues in the clear must were 0.02-0.04 mg L -1 . At the end of the wine-making process, flufenoxuron residues were not determined in the produced wines (Table 4) with both vinification techniques (with or without maceration) showing the same influence of vinification technique on flufenoxuron residue reduction.

Lufenuron. Lufenuron showed analogous to flufenoxuron persistence on grapes during the field experiments. During 2004, residues of lufenuron on grape samples at 0 DAA ranged from 0.39±0.03 to 0.46±0.09 mg kg -1 for "Cabernet" and "Roditis" grapes, respectively. Thereafter, lufenuron residues decreased very slowly with time to 0.38±0.03 and 0.39±0.04 mg kg -1 at 7 DAA, and to 0.31±0.04 and 0.28±0.02 mg kg -1 at 21 DAA for "Cabernet" and "Roditis" grapes, respectively. Thereafter, they decreased further to 0.26±0.01 and 0.25±0.03 mg kg -1 (representing 54-66% of the initial residues) at 42 DAA, which is the recommended by the manufacturers PHI for lufenuron. For 2006, grapes harvested at 40 DAA contained lufenuron residues at 0.27±0.03 and 0.24±0.01 mg kg -1 for "Roditis" and "Cabernet" cultivars, respectively. Residues of lufenuron in all our grape subsamples both in 2004 and 2006 were clearly below the recently established by EU (31/8/2008) MRL value (1.0 mg kg -1 ) for lufenuron on grapes even few hours after the treatment (at 0 DAA).

Lufenuron dissipation on grapes (Figure 3) showed pseudo-first-order kinetics (R 2 > 0.897) with low values of reduction rates ranging from 0.011 for "Cabernet" and 0.016 for "Roditis" grapes. Considering our data for flufenoxuron and lufenuron dissipation and the published data for teflubenzuron dissipation in grapes, between the three benzoylurea insecticides, teflubenzuron seems more persistent than the other two.

The grape samples used to study lufenuron residues partitioning during vinification process were harvested at 21 and 30 DAA for the wine-making process in 2004 and from 21 and 40 DAA for that in 2006. During wine production without maceration and before fermentation, a part of lufenuron residues was removed with the solid part of grapes and the remaining residues in must ranged from 0.09 to 0.16 mg kg -1 with mean values of transfer factor from grapes to must at 0.45 and 0.43, for "Roditis" and "Cabernet" grapes, respectively. After the must centrifugation, lufenuron residues in the clear must were 0.01-0.03 mg L -1 and the calculated transfer factor from must to wine was <0.08, indicating that lufenuron was strongly adsorbed by the suspended solids in the must. Wines obtained at the end of the wine-making process (with and without 13 maceration) were residue-free (below the method LOQ value of 0.01 mg L -1 ) and the calculated transfer factor for lufenuron from grape to wine was <0.04 for both "Cabernet" and "Roditis" grapes.

Tebufenozide. After treatment, at 0 DAA, mean concentration of tebufenozide residues on grapes were 0.74±0.04 to 0.95±0.14 mg kg -1 , for "Cabernet" and "Roditis" grapes, respectively. Tebufenozide residues during the experimental trial dissipated slowly according to pseudo-first-order kinetics (R 2 = 0.8809-0.8878) with reduction rates ranging from 0.011 for "Cabernet" to 0.018 for "Roditis" grapes (Figure 4). In particular, tebufenozide residues decreased slowly to 0.72±0.05 and 0.68±0.06 mg kg -1 at 7 DAA, then to 0.65±0.06 and 0.59±0.05 mg kg -1 at 14 DAA reaching 0.46±0.05 and 0.40±0.03 mg kg -1 at 42 DAA (representing 62 and 42% of the initially deposited amount on grapes) for "Cabernet" and "Roditis" grapes, respectively. Tebufenozide residues determined in grapes 21 DAA, that is the recommended PHI, ranged from 0.55±0.03 and 0.68±0.07 mg kg -1 , values clearly lower than the proposed MRL value for tebufenozide in grapes established by EU (3 mg kg -1 ) and Codex Alimentarium (2 mg kg -1 ).

The grape samples used to study tebufenozide partitioning during vinification process were harvested at 21 and 30 DAA for the wine making process in 2004 and at 21 and 40 DAA for that in 2006. Wine prepared with and without maceration from treated grapes of both cultivars contained tebufenozide residues ranging from 0.13 to 0.26 mg L -1 for both vinification processes. The observed concentration of tebufenozide residues in wines produced from grapes collected at or after the recommended PHI exceed the tolerance value established for wine by Switzerland (0.1 mg kg -1 ) but they are in accordance with the MRL value for tebufenozide in wine (0.3 mg L -1 ) proposed factors from grape to wine ranged from 0.45 for vinification of white 'Roditis' to 0.34 of red 'Cabernet' grapes for the wine-making process without maceration and from 0.31 to 0.32, respectively, for the wine-making process with maceration.

During wine production without maceration and before fermentation, residues were removed with the solid phase of grapes (Tables 1234) and the remaining residues in must were 0.17 to 0.33 mg kg -1 resulting in tebufenozide transfer factors from grape to must 0.48 for "Cabernet" and 0.52 for "Roditis", values almost similar to those observed for the other two insecticides studied. After the must centrifugation, tebufenozide residues in the clear must were 0.07-0.12 mg L -1 , indicating that tebufenozide was only partially adsorbed by the suspended solids of the must. The calculated mean values of transfer factor for tebufenozide from must to wine were 0.73 for "Cabernet" and 0.86 for "Roditis", values much higher than those calculated for flufenoxuron and lufenuron (<0.08). The observed transfer factors of tebufenozide from grapes into must, centrifuged must, and in wine in all cases indicated a similar pattern of tebufenozide fate for both cultivars of white 'Roditis' and red 'Cabernet' grapes.

The clearly different fate of tebufenozide residues relatively to those of flufenoxuron and lufenuron during the wine-making process is due mainly to their different distribution capacity between the liquid and solid phase of the produced must and wine as found from the calculated high tebufenozide transfer factor values from must to centrifuged must and from must to wine.

Wine clarification experiments performed for wines containing tebufenozide residues (Figure 5) showed that among the tested clarifying agents, i.e. bentonite, potassium caseinate, gelatine-silicon dioxide and polyvinylpolypyrrolidone did not presented significantly decrease the pesticide residue concentration compared with non- clarified wine. On the other hand, clarification with charcoal significantly reduced (by 95%) tebufenozide residues in wine confirming that it is the most effective between other clarifying agents for reducing various pesticides in wine [START_REF] Cabras | Fate of some insecticides from vine to wine[END_REF], Tsiropoulos et al. 1999[START_REF] Ruediger | Removal of pesticides from red and white wine by the use of fining and filter agents[END_REF], Fernandez et al. 2005b[START_REF] Oliva | Removal of famoxadone, fluquinconazole and trifloxystrobin residues in red wines. Effects of clarification and filtration processes[END_REF].

Conclusions

All of the 'insect growth regulator insecticides' studied showed very slow dissipation in grapes in the field described by pseudo-first-order kinetics and giving dissipation rate values between 0.011 and 0.018 d -1 . Their residues' concentrations at the recommended PHI were clearly below the established MRLs in grape by EU (2 mg kg -1 for flufenoxuron, 1 mg kg -1 for lufenuron and 3 mg kg -1 for tebufenozide), while the measured concentration were below the MRL value even just after application.

Therefore, the use of these insecticides should not create limit problems if used following good agricultural practices.

The processing of treated grapes into wine almost eliminated residues for flufenoxuron and lufenuron resulting to residues-free wine, whereas tebufenozide was found in wine at concentrations up to near the 1/10 of the MRL value in grape. The two wine-making techniques employed (with and without maceration) had the same influence on tebufenozide residues in produced wine. Among the clarifying agents Table 1. Flufenoxuron, lufenuron and tebufenozide residues from grapes (mg kg -1 *) to wine (mg L -1 **) for 'Roditis' grapes collected at different interval times (days after

  18 o C in glass vials. An intermediate mixture standard solution containing all compounds, at 100 mg L -1 each, was prepared in acetone from the individual stock solutions and stored at -18 o C. Spiking solutions were from the intermediate solution and stored at 4 o C. Calibration standard solutions at concentrations 0.05 to 10 mg L -1 (9 solutions) in acetonitrile-water (1/1, v/v) were prepared from the intermediate solution.

  grapes collected for each cultivar and each experimental plot at 21 and 30 DAA from the field trials of 2004 and at 21 and 40 DAA from those of 2006

  acetonitrile-water (55/45, v/v) over 10 min to stabilize the HPLC system before starting the next run, giving 35 min as total run time. The injection volume was 20 µL. Before injection, samples were filtered through Titan 2 HPLC nylon membrane filters (17 mm, 0.2 µm pore size). The optimum detection was obtained at 210 nm.Under these chromatographic conditions tebufenozide, flufenoxuron and lufenuron were well separated and their concentrations were determined by the external standard technique by comparing the peak heights in the samples with those found in the calibration solutions.

  1 shows chromatograms of a mixture standard solution as well as of extracts of grape sample and of the obtained wine sample from the field experiments.

  27±0.03 and 0.26±0.04 mg kg -1 , respectively. Concentration values of flufenoxuron residues measured on all grape subsamples collected few hours after treatment up to near the PHI are clearly below the recently established by EU (

  Organisation for Vine and Wine (OIV). The calculated transfer

  Figures captions

Figure 2 .

 2 Figure 2. Field dissipation of flufenoxuron residues in 'Roditis'(• ) and 'Cabernet Sauvignon' (▲ ---) grapes for 2004 and decline curves as derived from the 1 st -order kinetic model. The solid points show mean values of flufenoxuron residues and error bars represent 2xSD of three replicates.

Figure 3 .

 3 Figure 3. Field dissipation of lufenuron residues in 'Roditis'(• ) and 'Cabernet Sauvignon'(▲ ---) grapes for 2004 and decline curves as derived from the 1 st -order kinetic model. The solid points show mean values of lufenuron residues and error bars represent 2xSD of three replicates.

Figure 4 .

 4 Figure 4. Field dissipation of tebufenozide residues in 'Roditis'(• ) and 'Cabernet Sauvignon'(▲ ---) grapes for 2004 and decline curves as derived from the 1 st -order kinetic model. The solid points show mean values of tebufenozide residues and error bars represent 2xSD of three replicates.

Figure 5 .

 5 Figure 5. Mean % overall reduction of tebufenozide residues in wines produced from

  ) for 2004 experiments. Relative standard deviation of three replicates is shown in parenthesis

  Figure1

Table 2 .

 2 Flufenoxuron, lufenuron and tebufenozide residues from grapes (mg kg -1 *) to

	wine (mg L -1 **) for 'Cabernet Sauvignon' grapes collected at different interval
	times (days after application, DAA) for 2004 experiments. Relative standard
	deviation of three replicates is shown in parenthesis

Table 3 .

 3 Flufenoxuron, lufenuron and tebufenozide residues from grapes (mg kg -1 *) to

	wine (mg L -1 **) for 'Roditis' grapes collected at different interval times (days after
	application, DAA) for 2006 experiments. Relative standard deviation of three
	replicates is shown in parenthesis

Table 4 .

 4 Flufenoxuron, lufenuron and tebufenozide residues from grapes (mg kg -1 *) to

	wine (mg L -1 **) for 'Cabernet Sauvignon' grapes collected at different interval
	times (days after application, DAA) for 2006 experiments. Relative standard
	deviation of three replicates is shown in parenthesis

Table 1 .

 1 Flufenoxuron, lufenuron and tebufenozide residues from grapes (mg kg -1 *) to

	wine (mg L -1 **) for 'Roditis' grapes collected at different interval times (days after
	application, DAA) for 2004 experiments. Relative standard deviation of three replicates is
	shown in parenthesis.
		Flufenoxuron	Lufenuron	Tebufenozide
	DAA	21	30	21	30	21	30
	Grapes *	0.31 (14) 0.28 (11) 0.30 (10) 0.22 (12) 0.55 (11) 0.43 (15)
	Μust *	0.14 (10)	0.13 (9) 0.13 (12) 0.11 (16) 0.30 (14) 0.27 (17)
	Centrifuged	0.03 (12) 0.02 (13) 0.03 (11) 0.02 (15) 0.12 (12) 0.07 (14)
	Μust **
	Wine with	<LOQ	<LOQ	<LOQ	<LOQ	0.14 (9)	0.13 (10)
	maceration **
	Wine without	<LOQ	<LOQ	<LOQ	<LOQ	0.26 (12) 0.23 (12)
	maceration **

Table 2 .

 2 Flufenoxuron, lufenuron and tebufenozide residues from grapes (mg kg -1 *) to wine (mg L -1 **) for 'Cabernet' grapes collected at different interval times (days after application, DAA) for 2004 experiments. Relative standard deviation of three replicates is shown in parenthesis.

		Flufenoxuron	Lufenuron	Tebufenozide
	DAA	21	30	21	30	21	30
	Grapes *	0.24 (10) 0.23 (12) 0.31 (11) 0.27 (8)	0.68 (10) 0.56 (12)
	Μust *	0.13 (14) 0.11 (15) 0.16 (9) 0.12 (15) 0.27 (12) 0.33 (18)
	Centrifuged
	Μust **	0.03 (9)	0.02 (11)	0.01	0.02 (17)	0.08 (7)	0.11 (10)
	Wine with
	maceration **	<LOQ	<LOQ	<LOQ	<LOQ	0.21 (12)	0.17 (9)
	Wine without
	maceration **	<LOQ	<LOQ	<LOQ	<LOQ	0.23 (9)	0.19 (11)

Table 3 .

 3 Flufenoxuron, lufenuron and tebufenozide residues from grapes (mg kg -1 *) to wine (mg L -1 **) for 'Roditis' grapes collected at different interval times (days after application, DAA) for 2006 experiments. Relative standard deviation of three replicates is shown in parenthesis.

		Flufenoxuron	Lufenuron	Tebufenozide
	DAA	21	40	21	40	21	40
	Grapes *	0.32 (9)	0.26 (14) 0.33 (8) 0.27 (10) 0.69 (12)	0.35 (9)
	Μust *	0.17 (11) 0.12 (10) 0.14 (11) 0.12 (5)	0.30 (9)	0.17 (10)
	Centrifuged	0.03 (8)	0.02 (7) 0.02 (12)	0.01	0.09 (12)	0.07 (7)
	Μust **
	Wine with	<LOQ	<LOQ	<LOQ	<LOQ	0.19 (8)	0.14 (7)
	maceration **
	Wine without	<LOQ	<LOQ	<LOQ	<LOQ	0.24 (10) 0.16 (11)
	maceration **

Table 4 .

 4 Flufenoxuron, lufenuron and tebufenozide residues from grapes (mg kg -1 *) to wine (mg L -1 **) for 'Cabernet Sauvignon' grapes collected at different interval times (days after application, DAA) for 2006 experiments. Relative standard deviation of three replicates is shown in parenthesis.

		Flufenoxuron	Lufenuron	Tebufenozide
	DAA	21	40	21	40		40
	Grapes *	0.34 (9)	0.27 (11) 0.29 (8)	0.24 (6)	0.73 (12)	0.39 (9)
	Μust *	0.18 (8)	0.14 (7) 0.11 (10) 0.09 (9)	0.32 (11)	0.19 (7)
	Centrifuged	0.04 (12)	0.02 (9)	0.01	0.02 (10)	0.07 (5)	0.09 (10)
	Μust **
	Wine with	<LOQ	<LOQ	<LOQ	<LOQ	0.25 (8)	0.13 (6)
	maceration **
	Wine without	<LOQ	<LOQ	<LOQ	<LOQ	0.23 (5)	0.15 (9)
	maceration **

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Tsiropoulos, N.G., Aplada-Sarlis, P., and Miliadis, G.E. 1999. Evaluation of teflubenzuron residue levels in grapes exposed to field treatments and in the must and wine produced from them. Journal of Agricultural and Food Chemistry, 47: 4583-4586.

Vaquero-Fernandez L. Sanz-Ansensio J., Lopez-Alonso M., Martinez-Soria M.T. 2009.

Fate and distribution of pyrimethanil, metalaxyl, dichlofluanid and penconazole fungicides from treated grapes intended for winemaking. Food Additives and Contaminants,


Jérémy Lecoeur

Jack H Noble

Ramya Balachandran

Robert F Labadie

Benoit M Dawant

Variability of the temporal bone surface's topography: implications for otologic surgery

Keywords: temporal bone, bone topography, otologic surgery, robotic surgery, universal stereotactic frame

Otologic surgery is performed for a variety of reasons including treatment of recurrent ear infections, alleviation of dizziness, and restoration of hearing loss. A typical ear surgery consists of a tympanomastoidectomy in which both the middle ear is explored via a tympanic membrane flap and the bone behind the ear is removed via mastoidectomy to treat disease and/or provide additional access. The mastoid dissection is performed using a high-speed drill to excavate bone based on a pre-operative CT scan. Intraoperatively, the surface of the mastoid component of the temporal bone provides visual feedback allowing the surgeon to guide their dissection. Dissection begins in "safe areas" which, based on surface topography, are believed to be correlated with greatest distance from surface to vital anatomy thus decreasing the chance of injury to the brain, large blood vessels (e.g. the internal jugular vein and internal carotid artery), the inner ear, and the facial nerve. "Safe areas" have been identified based on surgical experience with no identifiable studies showing correlation of the surface with subsurface anatomy. The purpose of our study was to investigate whether such a correlation exists. Through a three-step registration process, we defined a correspondence between each of twenty five clinically-applicable temporal bone CT scans of patients and an atlas and explored displacement and angular differences of surface topography and depth of critical structures from the surface of the skull. The results of this study reflect current knowledge of osteogenesis and anatomy. Based on two features (distance and angular difference), two regions (suprahelical and posterior) of the temporal bone show the least variability between surface and subsurface anatomy.

DESCRIPTION OF PURPOSE

A large component of otologic surgery involves drilling away a portion of the temporal bone via a procedure known as a mastoidectomy. This operation is similar to an archeological dig where overlying bone is excavated and vital subsurface structures are intended to be identified without damage. In otologic surgery, these structures include the brain, large blood vessels (e.g. the intracranial continuation of the internal jugular vein), nerves (e.g. the facial nerve which controls movement of the face) and the inner ear. During surgical training, surgeons are taught to begin a mastoidectomy in the middle of surface landmarks. The superior surface landmark is a boney ridge that is the continuation of the zygomatic arch (a.k.a. cheekbone) called the temporal line which lies roughly perpendicular to the superior aspect of the root of the ear. A more practical approximation is the posterior projection of straight temple eyeglass pieces. The anterior surface landmark is the most superficial aspect of the external auditory canal. The posterior landmark is considered most variable and consists of a hole in the bone through which a blood vessel called the emissary vein courses. The mid-region of this triangle, called Macewen's triangle [START_REF] Pickard | A Re-Examination of Macewen's Triangle as a Surface Marking for the Mastoid Antrum[END_REF][START_REF] Macewen | Pyogenic and infectious Diseases of the Brain and Spinal Cord[END_REF] (Figure 1), is anecdotally considered to be the safest location to begin surgical intervention. We sought to substantiate this anecdotal finding as future otological surgical techniques, particularly imageguided, minimally-invasive approaches, may have higher dependence on the reliability of surface topography.

One such intervention where this analysis of temporal bone surface topography would be useful is cochlear implantation, a surgery in which hearing loss is treated by inserting an electrode array into the cochlea. Traditional cochlear implantation procedures require wide excavation of the mastoid region of the temporal bone [START_REF]Food and Drug Administration[END_REF] to allow surgeons to visualize the critical subsurface structures. Over the last several years, we have developed a less-invasive, image-guided cochlear implantation procedure referred to as percutaneous cochlear implantation (PCI) [START_REF] Labadie | Clinical Validation Study of Percutaneous Cochlear Access using patient-Customized Microstereotactic Frames[END_REF] . In PCI, access to the cochlea is achieved by drilling via a linear path from the surface of the skull through the facial recess to the cochlea. Along the path is the facial recess region that is about 2.4 mm wide and is bounded by the facial nerve, which if damaged causes ipsilateral facial paralysis, and the chorda tympani, which if damaged causes ipsilateral loss of taste sensitivity. To achieve PCI accurately without damage to vital structures, mainly the facial nerve, we employ image-guided surgical (IGS) technology and use three bone-implanted anchors as fiducial markers and a customized microstereotactic frame that mounts on these anchors and constrains a drill along the desired path. The desired path is computed to be probabilistically safe with respect to sensitive structures 5 , which are segmented using automated techniques we have developed 6,7,8 The three bone-implanted fiducial anchors are commonly configured with one in the mastoid tip region, one in the suprahelical region, and one in a posterior region. Through our experience with PCI, we identified that it is necessary to attach the anchors at particular locations in these regions in order to design a suitable microstereotactic frame to achieve the desired trajectory. Currently we are in the process of designing a bone-attached robot [START_REF] Kratchman | Design of a Bone-Attached Parallel Robot for Percutaneous Cochlear Implantation[END_REF] for the drilling task (a prototype of the robot is visible on Figure 2). For this robot, we need to define a universal frame for mounting the robot easily on every patient. Such a fixed robotic frame calls for fixed angle of each leg of the frame with respect to the surface of the bone to enable rigid attachment of the frame and robot to the bone and for fixed lengths between the anchor points. To define the locations of the anchors onto which the fixed robot frame will be mounted, we designed this study to explore the variability of the surface of the temporal bone region around the ear canal in order to find the least varying areas in both displacement and angle amongst the patients.

METHODS

Images

Clinically-applicable temporal bone CT scans of twenty five patients were used in this study with various field of view, resolution, quality, and orientation. A typical scan is 768 × 768 × 205 voxels with, approximately, 0.215 × 0.215 × 0.336 mm 3 voxel size. Another CT scan was used as the reference (atlas) volume. This scan is 768 × 768 × 145 voxels with 0.21875 × 0.21875 × 0.4 mm 3 voxel size.

Registration procedure

The temporal bone of each patient was registered to the atlas using a three-step process. (1) The patient CT and the atlas were rigidly registered globally using an intensity-based registration algorithm based on normalized mutual information [START_REF] Studholme | An overlap invariant entropy measure of 3D medical image alignment[END_REF] . Figure 3 shows the result of this registration. After this first registration has been computed, both the patient CT and atlas images were cropped around the ear canal. The size of this new and smaller image is 44.625 x 61.25 x 50 mm 3 . This region represents the zone of the skull where the anchors would be implanted for all the PCI patients. It is thus a realistic zone of surgical action. ( 2) The cropped region of the patient CT and atlas were then rigidly registered again using the same algorithm and similarity measure.

Leg of the frame

This second registration provides a finer alignment of the temporal bone region of each patient to the atlas as shown on Figure 4. (3) Finally, the rigidly aligned cropped regions were registered using a non-rigid registration based on the adaptive bases algorithm [START_REF] Rohde | The Adaptive Bases Algorithm for Intensity-Based Nonrigid Image Registration[END_REF] . Figure 5 shows the result of this registration. Figure 6 shows the registration results of five patients. We see that the final result of the registration is visually correct.

To further explore the validity of the transformation, we looked closely at the chorda tympani, a small branch of the facial nerve which courses through pneumatized bone. Figure 7 shows that the tubular shape of this structure is preserved and that the chordae are aligned well despite their small size (about 11 mm long and 0.5 mm large).

Displacement and differential orientation maps

We were interested in knowing the variability of the temporal bone surface and quantified it using displacement and differential orientation maps among patients. Displacement is measured as the magnitude of the deformation field from the atlas to each of the patient images after the two rigid registrations. The mean and standard deviation of these displacements were then calculated and projected on the mesh of the atlas bone to produce displacement maps. The deformation fields also allow us to pair corresponding points on the surface of the bone.

Another crucial factor in the placement of bone-implanted fiducial anchors is the normal to the surface as the screws of the anchors need to be implanted perpendicularly to the bone to ensure rigid attachment. For each patient and each point in the surface of the cropped region after the two rigid registrations, we computed the outward normal to the surface. The cropped images after the second rigid registration were used because the temporal bones of all patients are locally aligned and the orientation is the same. The normal was also computed for the atlas image. We then computed the angle between the normals for each pair of images (atlas vs. patient). We then again computed the average and standard deviation of these angles to produce differential orientation maps.

Depth of intracranial structures

In order to quantify the degree with which Macewen's triangle can be considered safe, we need to know how deep from the surface of the skull are the intracranial structures that otological surgeons need to avoid, namely the cochlea, the chorda tympani, the facial nerve, the sigmoid sinus and the carotid artery.

Using the algorithms we have developed for this purpose [START_REF] Noble | Automatic determination of optimal linear drilling trajectories for cochlea access accounting for drill-positioning error[END_REF][START_REF] Noble | Automatic segmentation of the facial nerve and chorda tympani in CT images using spatially dependent feature values[END_REF][START_REF] Noble | Automatic Identification and 3-D Rendering of Temporal Bone Anatomy[END_REF] , we segmented those five structures in each of the 25 patients. We were interested in the distance of each structure from the skull as this distance will be the most useful for surgeons as they approach targets following the right-left axis of the head. Using the algorithm developed by Prima et al. [START_REF] Prima | Computation of the Mid-Sagittal Plane in 3D Brain Images[END_REF] , we computed the mid-sagittal plane in each of the patients and we defined the right-left axis as the normal to that plane. We then computed the distance between the surface of the skull and each of the five structures along that direction. Figure 8 shows a map of the depth of these structures for a patient. Whenever two structures lied on the path from the skull surface point following the right-left axis, we kept the minimal distance on those maps. Using the registration field defined in the previous sub-section, we registered these maps to the atlas and we then computed the average distance and standard deviation for each skull surface point.

RESULTS

Displacement and differential orientation maps

Figures 9 and 10 show the average and standard deviation of the displacement respectively. The average displacement ranges from 1.36 mm to 5.23 mm while the standard deviation ranges from 0.38 mm to 3.51 mm. Regions usually used to attach the anchors for PCI are labeled in Figure 9 with regions 1 and 3 indicating the suprahelical regions, region 2 indicating the mastoid region, and region 4 indicating the posterior region. The portion of the temporal bone directly above the auditory canal, labeled 1 in Figure 9, exhibits the least displacement, both in average and standard deviation, while the mastoid, labeled 2, is the region of the temporal bone where the displacement is the largest with the largest standard deviation. The average angle between normals of corresponding points of the surface of the bone ranges from 9.5 to 44.3 degrees while standard deviation ranges from 4.7 to 33.5 degrees. Figure 11 and 12 illustrate those features on the surface of the temporal bone region. Based on both the measured features, we can see that regions labeled 1 and 4 exhibit low displacement and low differences in normal orientation. These two zones are very good candidates for bone-implanted, fiducial anchor placement.

Regions labeled 2 and 3 exhibit large displacements. They also correspond to muscle attachments: the sternocleidomastoid muscle for region 2 and the auricularis posterior muscle for region 3. These muscle attachments tend to pull on the bone, hence creating protrusions during osteogenesis.

Depth of intracranial structures

Figure 13 present the average distance of intracranial structures from the surface of the skull along the right-left axis of the head. In Macewen's triangle region, this distance ranges from 17.8 to 38.3 mm. Figure 14 provides the standard deviation of the distance from the surface of the skull of those same structures. We observer that this standard deviation is relatively low in Macewen's triangle as it ranges from 0.6 to 7.2 mm. The highest standard deviation is obtained where the depth is between 35 and 38 mm, hence representing less that 20% variation, and only in a small portion of the area..

DISCUSSION

To the best of our knowledge, this work reports the first topography study of the temporal bone. Using a combination of two rigid-body and one non-rigid transformation, we were able to compare the temporal bones of twenty five patients and show how they differ. From this comparison, we were then able to define two regions of the skull surface (suprahelical and posterior) exhibiting low variation of displacement and angle that are good candidates for anchor placement. We were also able to link the highly variable regions to a muscle attachment locations. This work also shows that the mastoid region is highly variable, indicating a difficulty in defining a common location for the third anchor.

Using the atlas space as common space for registration is a potential bias if the atlas is not representative of the population. To tackle that problem, it would be good to reiterate the whole process using an average of the 25 volumes as the new atlas. Moreover, separate atlases may be necessary for specific populations such as children.

We also designed a process to compute the depth of important intracranial structures (chorda tympani, facial nerve, cochlea, sigmoid sinus and carotid artery) from the surface of the skull from the point of view of an otological surgeon.

From this measurement, we established that the traditional training, which consider Macewen's triangle to be a safe location to begin intervention, is correct as critical structure lies between 17.8 and 38.3mm deep from that region, hence giving a relatively large margin of safety when beginning to drill.. A limitation of this part of the study is that we did not compute the depth of the brain from the surface of the skull, which infrequently is located inferior to the temporal line (the superior aspect of Macewen's triangle); we are currently addressing this limitation by incorporating the brain into our atlas.

CONCLUSION

This study explored the topography of the temporal bone and its implications for otologic surgery. Regarding identification of sites for bone-implanted, fiducial anchor placement, we identified two regions of low inter-patient variability-suprahelical (region 1 in Figure 9) and posterior (region 4 in Figure 9)-which would be good candidates for standard anchor placement. We also discovered that a third standard anchor location may be difficult to find as the traditional third region-mastoid tip (region 2 in Figure 9)-shows high inter-patient variability. We also show that the intracranial structures that otologic surgeries try to avoid are deep enough within Macewen's triangle to confirm that the traditional drilling approach is safe.

Figure 1 -

 1 Figure 1 -Macewen's Triangle for a left ear of a patient is indicated by the region bounded by the three lines-temporal line (solid line), posterior edge of the external auditory canal (dashed line), and the line that connects the other two lines (dotted line).

Figure 2 -

 2 Figure 2 -Prototype of a PCI robot with its fixed frame (black part) mounted on a temporal bone. The frame is rigidly attached to the bone using anchors.

Figure 3 -

 3 Figure 3 -First step of registration: rigid registration of whole volume -(A) Patient CT scan; (B) atlas; (C) patient scan after registration; (D) correspondence between patient after registration (in blue) and atlas (in grey).

Figure 4 -

 4 Figure 4 -Second step of registration: rigid registration of subvolumes -(A) Patient CT scan; (B) atlas; (C) patient scan after registration; (D) correspondence between patient after registration (in blue) and atlas (in grey).

Figure 5 -

 5 Figure 5 -Final step of registration: non-rigid registration of subvolumes -(A) Patient CT scan; (B) atlas; (C) patient scan after registration; (D) correspondence between patient after registration (in blue) and atlas (in grey).

Figure 6 -

 6 Figure 6 -Example of registration results. (a) to (e): temporal bone from five different patients. (f) to (j) temporal bone of the same patient after registration to the atlas. (k) atlas.

Figure 7 -

 7 Figure 7 -Visualization of internal structures after the three-step registration. Left: chorda tympani of five patients registered and transformed to the atlas with the temporal bone shown as the transparent structure. Right: magnification of the chordae tympani showing preservation of their shape and good alignment.

Figure 8 -

 8 Figure 8 -Depth map of intracranial structure of a patient

Figure 9 -Figure 10 -

 910 Figure 9 -Average displacement map of the temporal bone

Figure 11 -Figure 12 -

 1112 Figure 11 -Mean differential orientation map of the temporal bone

Figure 13 -

 13 Figure 13 -Average distance of intracranial structures from the surface of the skull-Left: temporal bone -Right: Magnification of Macewen's triangle region (dotted line). Color map from 0 to 50 mm.

Figure 14 -

 14 Figure 14 -Standard deviation of the distance of intracranial structures from the surface of the skull -Left: temporal bone -Right: Magnification of Macewen's triangle region (dotted line). Color map from 0 to 8.33 mm.

ACKNOWLEDGMENTS

This project is supported, in parts, by NIH grants R01DC008408 and R01DC010184 from the National Institute of Deafness and Other Communication Disorders. The content is solely the responsibility of the authors and does not necessarily represent the official views of these institutions.


S P Van Tongeren
email: s.p.van.tongeren@med.umcg.nl

J E Degener

H J M Harmsen

Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR

Keywords: Rapid, easy, DNA extraction, FTA, qrtPCR, Escherichia coli, Staphylococcus aureus

The development of fast and easy on-site molecular detection and quantification methods for hazardous microbes at solid surfaces is desirable for several applications where specialised laboratory facilities are absent. Quantification of bacterial contamination necessitates the assessment of the efficiency of the used methodology as a whole, including the preceding steps of sampling and sample processing. We used quantitative real-time PCR (qrtPCR) for Escherichia coli and Staphylococcus aureus to measure the recovery of DNA from defined numbers of bacterial cells that were subjected to three different DNA extraction methods: the QIAamp ® DNA Mini Kit, Reischl et al. method and FTA ® Elute. FTA ® Elute significantly showed the highest median DNA extraction efficiency of 76.9 percent for E. coli and 108.9 percent for S. aureus. The Reischl et al. method and QIAamp ® DNA Mini Kit inhibited the E. coli qrtPCR assay with a 10-fold decrease of detectable DNA. None of the methods inhibited the S. aureus qrtPCR assay. The FTA ® Elute applicability was demonstrated with swab samples taken from the International Space Station (ISS) interior. Overall the FTA ® Elute method was found most suitable to selected criteria in terms of rapidity, easiness of use, DNA extraction efficiency, toxicity and transport and storage conditions.

Introduction

The detection and quantification of microbes present on solid surfaces plays an important role in clinical settings, the food industry, drinking water distribution systems, air-conditioning systems in modern buildings or advanced life-support systems, such as manned spacecraft. Detection and quantification by conventional culturing techniques however is laborious, lengthy, requires specialist expertise, has poor diagnostic sensitivity and in addition, many bacteria such as Faecalibacterium prausnitzii are difficult to culture or uncultivable which hampers their detection [START_REF] Zengler | Accessing uncultivated microorganisms: from the environment to organisms and genomes and back[END_REF]. Moreover, the culturing of hazardous microbes such as pathogens and so-called "technophiles" may often be undesirable [START_REF] Van Tongeren | Microbial detection and monitoring in advanced life support systems like the International Space Station[END_REF]. Especially in remote locations such as field hospitals, disaster areas or manned spacecraft where specialised laboratory facilities and technical personnel are not available, this results in the need for fast and easy methodologies that can be used on-site by non-specialists. Accordingly, molecular methods have increasingly been used for the detection and quantification of hazardous microbes overcoming many of the disadvantages encountered by culturing [START_REF] Espy | Real-time PCR in clinical microbiology: applications for routine laboratory testing[END_REF].

Quantitative real-time PCR (qrtPCR) is a cultivation-independent method for the rapid molecular detection and quantification of nucleic acid sequences [START_REF] Espy | Real-time PCR in clinical microbiology: applications for routine laboratory testing[END_REF]. The method has a high specificity with a detection sensitivity of a few molecules per reaction and the potential to be automated and miniaturized for use on-site by non-specialists. Moreover, for a reliable quantification of bacterial contamination it is important to assess the efficiency of the used methods as a whole, including both the molecular detection method as well as the preceding steps of sampling and sample processing [START_REF] Espy | Real-time PCR in clinical microbiology: applications for routine laboratory testing[END_REF][START_REF] Bastien | Quantitative real-time PCR is not more sensitive than "conventional" PCR[END_REF][START_REF] Cook | Optimization of methods for detecting Mycobacterium avium subsp. paratuberculosis in environmental samples using quantitative, real-time PCR[END_REF]. In addition, for increased sensitivity, methods with the highest possible efficiency should be utilized and the dilution factor in all steps of sampling and sample processing kept to a minimum. Sample processing for use with qrtPCR involves cell lysis and subsequent recovery of DNA free of amplification inhibitors. Factors that play a role in the efficiency of bacterial cell lysis may be physiological characteristics of the species such as the constitution of the cell wall, the physiological state the cell is in or cell concentration [START_REF] Rantakokko-Jalava | Optimal DNA isolation method for detection of bacteria in clinical specimens by broad-range PCR[END_REF][START_REF] Ludwig | How quantitative is quantitative PCR with respect to cell counts?[END_REF][START_REF] Coyne | Comparative analysis of the Schleicher and Schuell IsoCode Stix DNA isolation device and the Qiagen QIAamp DNA Mini Kit[END_REF]. As a consequence, most DNA extraction methods are optimal for just one or a group of bacterial species [START_REF] Rantakokko-Jalava | Optimal DNA isolation method for detection of bacteria in clinical specimens by broad-range PCR[END_REF]. Ideally, for applications where it is essential to measure the levels of a variety of hazardous species such as in a natural environment, a universal DNA extraction method would be preferred that is optimal for all bacterial species.

The aim of this study was to select and validate a suitable DNA extraction method for the recovery of DNA from the potential pathogens Escherichia coli and Staphylococcus aureus to be used in qrtPCR. These species were used as model organisms as the gram-negative bacterium E. coli lyses differently than the gram-positive bacterium S. aureus, due to a different constitution of its cell wall. In particular for use on-site with samples taken from solid surfaces, we sought to select a fast and easy to use DNA extraction method that has a high efficiency, is universally effective for bacterial species, makes use of non-toxic chemicals and has little or no transport or storage requirements.

Materials and methods

Cultivation of bacterial cells

S. aureus subsp. aureus ATCC 25923 and E. coli ATCC 11775

T cells were each inoculated into 9 ml Brain Heart Infusion (BHI) (Mediaproducts BV, Groningen, The Netherlands) and grown overnight at 37°C. From each culture, cells were washed twice by centrifugation of 1 ml cell suspension for 10 min at 16100 × g, removal of the supernatant and resuspension of the pellet in 1 ml sterile physiological salt solution (0.85% NaCl, Mediaproducts BV, Groningen, The Netherlands). The suspensions were kept on ice during the remainder of the procedure to minimize cell growth and lysis.

DNA extraction methods description

With the commonly used QIAamp ® DNA Mini Kit (QIAGEN GmbH, Hilden, Germany) total DNA can be purified from a variety of biological, clinical and forensic specimens. The method uses chemical lysis by undisclosed lysis buffers containing chaotropic salt, enzymatic digestion by treatment with proteinase K, and thermal lysis. The method uses a separate protocol for Grampositive bacteria, with the additional use of enzymatic digestion by lysozyme or lysostaphin with

DNA extraction method comparison

The experiments were performed using DNA-free laboratory techniques. The cells in both E. coli and S. aureus suspensions were enumerated by 4",6-diamidino-2-phenylindole (DAPI) staining and subsequent visual cell counting with an Olympus BH2 epifluorescence microscope by the method as described previously [START_REF] Harmsen | Extensive set of 16S rRNA-based probes for detection of bacteria in human feces[END_REF], with the exception that the cells were not fixed by paraformaldehyde. The enumerations were performed in duplicate and averaged. Subsequently, the cell suspensions were serially diluted in sterile physiological salt solution and divided into aliquots of 5.3 × 10 To demonstrate the applicability of the FTA ® Elute method for the analysis of surface samples, DNA was extracted with this method from triplicate swab samples taken from the interior of the International Space Station (ISS) as part of the "SAMPLE" experiments which were previously described in van Tongeren et al. 2007 [START_REF] Van Tongeren | Microbial detection and monitoring in advanced life support systems like the International Space Station[END_REF]. The DNA contents were subsequently quantified with the qrtPCR assays of S. aureus and E. coli as described below.

Quantitative real-time PCR methods

Two TaqMan PCR assays were used to amplify specific regions of the genome of S. aureus and E. coli. Oligonucleotide primers and double-dye probes were obtained from Eurogentec S. A.

(Seraing, Belgium). Primers SauV58 (5"-GCTGTGATGGGGAGAAGACAT-3") and SauR54b (5"-CGGTACGGGCACCTATTTTC-3") were used to amplify a 90 bp fragment of the 23S rRNA gene of S. aureus [START_REF] Ludwig | How quantitative is quantitative PCR with respect to cell counts?[END_REF]. Primers UIDA-F (5"-TGGTGATTACCGACGAAAAC-3") and UIDA-R (5"-GCGTGGTTACAGTCTTGC-3") were used to amplify a 145 bp fragment of the uidA gene of E.

coli [START_REF] Brasher | Detection of microbial pathogens in shellfish with multiplex PCR[END_REF]. Probes Sta59bTQ (5"-AGAGGCTTTTCTCGGCAGTGTGAAATCAACGA-3") and UIDA-Pr (5"-GCCGGGATCCATCGCAGCGTAATGCTC-3") were hybridized in real-time with the PCR product of S. aureus and E. coli, respectively [START_REF] Ludwig | How quantitative is quantitative PCR with respect to cell counts?[END_REF][START_REF] Brasher | Detection of microbial pathogens in shellfish with multiplex PCR[END_REF]. , of the Sta59bTQ probe 80 nmol l -1 and of the UIDA-Pr probe 300 nmol l -1 . Amplification and detection were carried out on a Smart Cycler ® System (Cepheid, Sunnyvale, USA) with a profile of 50°C for 2 min to activate Uracil-N-Glycosylase, 95°C for 10 min to activate HotGoldStar DNA polymerase, followed by 45 cycles of 95°C for 15 s and 60°C for 60 s.

Negative controls for each qrtPCR assay were performed by running reactions without the addition of template DNA.

Generation of qrtPCR standards with bacterial genomic DNA Cells were cultivated as described above. To obtain a sufficient amount of relatively good quality DNA, the commonly used QIAamp ® DNA Mini Kit was used to extract DNA from the cells according to the instructions of the manufacturer. The procedure was followed by ethanol precipitation to purify the DNA solution from contaminants such as PCR inhibiting substances.

The DNA purity and concentration was determined with a NanoDrop ® ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). A standard curve of mean qrtPCR Ct values was prepared with triplicate replicates of serial dilutions of bacterial genomic DNA (three series of 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg and 10 fg for S. aureus subsp.

aureus ATCC 25923 and two series of 30 ng, 3 ng, 300 pg, 30 pg, 3 pg, 300 fg and 30 fg for E.

coli ATCC 11775 T , respectively). The amount of bacterial DNA in samples that were subjected to the various DNA extraction methods was estimated by qrtPCR with this curve under identical PCR conditions.

Statistical analysis

The quantification limit was determined for each qrtPCR assay as the lowest concentration on the linear part of the standard curve at which 7 out of 9 reactions or more gave a positive result. The quantification limit was used as the cut-off level or, instead of this, the value of a negative control in case it had a positive result. In case a Ct of zero (negative result) or above the mean Ct (+ 1 s.

d.) of the quantification limit was measured, a Ct equal to the mean Ct (+ 1 s. d.) of the quantification limit was assumed for data analysis.

Results

The processing time for 42 samples was 40 minutes for the

Discussion

Molecular methods have increasingly been used for the detection and quantification of hazardous microbes. Moreover, the need for a rapid and easy to use on-site molecular detection and quantification method is imperative. An equally important step in nucleic-acid based quantification of bacteria is the part of sample processing preceding the actual measurement, as has previously been mentioned [START_REF] Espy | Real-time PCR in clinical microbiology: applications for routine laboratory testing[END_REF][START_REF] Bastien | Quantitative real-time PCR is not more sensitive than "conventional" PCR[END_REF][START_REF] Cook | Optimization of methods for detecting Mycobacterium avium subsp. paratuberculosis in environmental samples using quantitative, real-time PCR[END_REF]. The results of this study confirm that DNA extraction is a key step in this process.

Several strategies can be used for the extraction of DNA from microbial samples, such as enzymatic, chemical or thermal lysis, mechanical disruption of the cell wall by beads or sonication or a combination of the above [START_REF] Rantakokko-Jalava | Optimal DNA isolation method for detection of bacteria in clinical specimens by broad-range PCR[END_REF][START_REF] Reischl | Rapid identification of methicillin-resistant Staphylococcus aureus and simultaneous species confirmation using real-time fluorescence PCR[END_REF]. A disadvantage of enzymatic lysis is that commercially available enzymes can be contaminated with microbial DNA. Highly sensitive and specific nucleic acid-based methods for the detection of bacteria necessitate the use of DNA extraction reagents that are free of contaminating bacterial nucleic acids. In addition, the use of enzymes often requires special conditions and preservation requirements such as refrigeration and buffer storage. Chemical lysis often involves the usage of aggressive and toxic chemicals which is less desirable for on-site detection where laboratory safety conditions are absent. A disadvantage for application on-site to thermal lysis, mechanical disruption by beads, or methods that use centrifugation is that the required equipment generally takes up a lot of weight and volume.

We selected to study three different DNA extraction methods to meet criteria for use on-site with qrtPCR on samples taken from solid surfaces such as processing time, easiness of use, efficiency, range of bacterial species that it is effective for, chemical toxicity and transport and storage requirements, which will be discussed below.

In our study, both the Reischl et al. protocol and FTA ® Elute were indeed fast and easy to use by single step isolation procedures and processing times of 40 and 110 minutes for 42 samples, respectively. DNA extraction methods with comparable processing times were reported before [START_REF] Espy | Real-time PCR in clinical microbiology: applications for routine laboratory testing[END_REF][START_REF] Hogg | Rapid detection of methicillinsusceptible and methicillin-resistant Staphylococcus aureus directly from positive BacT/Alert ® blood culture bottles using real-time polymerase chain reaction: evaluation and comparison of 4 DNA extraction methods[END_REF][START_REF] Cremonesi | Technical note: Improved method for rapid DNA extraction of mastitis pathogens directly from milk[END_REF][START_REF] Cenci-Goga | Detection of tet(M) gene from raw milk by rapid DNA extraction followed by a two-step PCR with nested primers[END_REF].

In all, FTA ® Elute paper significantly had the highest efficiency to recover DNA from E. coli DNA extraction efficiency is limited by several factors. An important factor is cell concentration, which can be a delimiting factor for quantification of natural samples where cell concentrations will vary. At low concentrations, method sensitivity will play a role and at high concentrations, overloading of the method can reduce DNA extraction efficiency. For FTA ® Elute problems can arise for certain harder to lyse species at concentrations above 10 7 cells ml -1 and overloading of the column of the QIAamp ® DNA Mini Kit may lead to significantly lower yields than expected, according to the manufacturers instructions. A second factor is the type of bacterial species that is being subjected to the extraction. For instance, the constitution of the bacterial cell wall may impede cell lysis and the liberation of DNA from the cell. A third factor may be the physiological state of the cells that are subjected to cell lysis [START_REF] Ludwig | How quantitative is quantitative PCR with respect to cell counts?[END_REF], in which context it can be noted that the cells used in the present study were of liquid cultures in stationary phase. Another factor that may influence the outcome of quantification is the size of the DNA fragments generated by a specific method [START_REF] Rantakokko-Jalava | Optimal DNA isolation method for detection of bacteria in clinical specimens by broad-range PCR[END_REF]. Long fragment sizes generated by the DNA extraction method may result in incomplete denaturation during the thermal cycling of qrtPCR, whilst on the other hand short fragment sizes may result in reduced amplification efficiency.

The QIAamp ® DNA Mini Kit was developed and commonly used for a variety of bacterial species [START_REF] Gullsby | Performance of Bordetella pertussis IS481 real-time PCR in a vaccine trial setting[END_REF][START_REF] Aldous | Comparison of six methods of extracting Mycobacterium tuberculosis DNA from processed sputum for testing by quantitative real-time PCR[END_REF][START_REF] Lee | A multiplex real-time PCR for differential detection and quantification of Salmonella spp., Salmonella enterica serovar Typhimurium and Enteritidis in meats[END_REF][START_REF] Luan | Use of activated carbon coated with bentonite for increasing the sensitivity of pcr detection of Escherichia coli O157:H7 in Canadian oyster (Crassostrea gigas) tissue[END_REF][START_REF] Wilson | Detection of Legionella pneumophila by real-time PCR for the mip gene[END_REF][START_REF] Wilson | Comparison of five methods for extraction of Legionella pneumophila from respiratory specimens[END_REF][START_REF] Peters | Quantitative detection of Staphylococcus aureus and Enterococcus faecalis DNA in blood to diagnose bacteremia in patients in the intensive care unit[END_REF][START_REF] Morio | Real-time PCR assay for the detection and quantification of Legionella pneumophila in environmental water samples: utility for daily practice[END_REF][START_REF] Simms | Associations between Mycoplasma genitalium, Chlamydia trachomatis, and pelvic inflammatory disease[END_REF][START_REF] Dauphin | Comparison of five commercial DNA extraction kits for the recovery of Yersinia pestis DNA from bacterial suspensions and spiked environmental samples[END_REF]. The FTA technology concept, including FTA ® Elute, was also developed for use with a broad spectrum of bacterial species [START_REF] Rajendram | Long-term storage and safe retrieval of DNA from microorganisms for molecular analysis using FTA matrix cards[END_REF][START_REF] Jaravata | Detection of Mycobacterium avium subsp. paratuberculosis in bovine manure using Whatman FTA card technology and Lightcycler real-time PCR[END_REF][START_REF] Nechvatal | Fecal collection, ambient preservation, and DNA extraction for PCR amplification of bacterial and human markers from human feces[END_REF]. To our knowledge, the use of FTA ® Elute for quantitative bacterial applications has not been assessed to date. The protocol by Reischl et al.

was previously used for S. aureus, however the main constituents and boiling procedure of the method have also been used for other species [START_REF] Reischl | Rapid identification of methicillin-resistant Staphylococcus aureus and simultaneous species confirmation using real-time fluorescence PCR[END_REF][START_REF] Wilson | Detection of Legionella pneumophila by real-time PCR for the mip gene[END_REF][START_REF] Reischl | PCR-based detection of mycobacteria in sputum samples using a simple and reliable DNA extraction protocol[END_REF][START_REF] Miller | Comparison of conventional susceptibility testing, penicillin-binding protein 2a latex agglutination testing, and mecA real-time PCR for detection of oxacillin resistance in Staphylococcus aureus and coagulase-negative Staphylococcus[END_REF][START_REF] Sakai | Simultaneous detection of Staphylococcus aureus and coagulase-negative staphylococci in positive blood cultures by real-time PCR with two fluorescence resonance energy transfer probe sets[END_REF][START_REF] Shrestha | Evaluation of the LightCycler Staphylococcus M GRADE kits on positive blood cultures that contained gram-positive cocci in clusters[END_REF][START_REF] Shrestha | Detection and differentiation of Mycobacterium tuberculosis and nontuberculous mycobacterial isolates by real-time PCR[END_REF][START_REF] Shrestha | Rapid identification of Staphylococcus aureus and the mecA gene from BacT/ALERT blood culture bottles by using the LightCycler system[END_REF]. However, as confirmed by our results, it must be taken into account that inter-species and even intra-species differences in DNA extraction efficiencies will exist, influencing the outcome of microbial quantification [START_REF] Rantakokko-Jalava | Optimal DNA isolation method for detection of bacteria in clinical specimens by broad-range PCR[END_REF][START_REF] Coyne | Comparative analysis of the Schleicher and Schuell IsoCode Stix DNA isolation device and the Qiagen QIAamp DNA Mini Kit[END_REF][START_REF] Zucol | Real-time quantitative broad-range PCR assay for detection of the 16S rRNA gene followed by sequencing for species identification[END_REF].

Furthermore, in contrast to FTA ® Elute, both the Reischl et al. and QIAamp ® DNA Mini Kit method showed inhibitory effects on the E. coli qrtPCR assay but not on the S. aureus qrtPCR assay.

Whilst the final volume of the FTA ® Elute extract is four times more dilute than that of the other methods, the concentration of any inhibitory compounds it may possibly contain is too low to cause an inhibitory effect when used according to the manufacturer"s instructions. As such, inhibitory effects of a DNA extraction method can differ per PCR assay as shown by comparison of our results and those of other authors [START_REF] Coyne | Comparative analysis of the Schleicher and Schuell IsoCode Stix DNA isolation device and the Qiagen QIAamp DNA Mini Kit[END_REF][START_REF] Aldous | Comparison of six methods of extracting Mycobacterium tuberculosis DNA from processed sputum for testing by quantitative real-time PCR[END_REF]. PCR inhibition reduces sensitivity, thereby negatively influencing the outcome of quantitative measurements. As a result, different rates of PCR inhibition on separate PCR assays will complicate quantification of microbial samples with a complex composition. Moreover, these observations should also be carefully considered in the choice of inhibition controls [START_REF] Espy | Real-time PCR in clinical microbiology: applications for routine laboratory testing[END_REF]. In addition to PCR inhibition generated by the DNA extraction method itself as described above, PCR inhibition needs to be assessed depending on the specimen type. In this study, we sought to select a DNA extraction method suitable for the processing of samples taken from solid surfaces in various surroundings. However, a wide variety of possible PCR inhibiting compounds or, most often, none at all can be emphasized to be present on such surfaces, leaving any prediction on PCR inhibition generated by this particular specimen type speculative.

The least toxic method appears to be FTA Finally, the applicability of the FTA ® Elute method for the analysis of environmental samples was demonstrated with swab samples taken from the interior of the ISS. The bacterial levels that correspond with the amount of DNA measured in these samples are also in agreement with hygiene measures taken aboard the ISS aimed to keep bacterial levels below the bacterial acceptability limit of 10 4 colony forming units per 100 cm 2 [START_REF] Pierson | Microbial contamination of spacecraft[END_REF].

The importance of the DNA extraction step in nucleic acid based molecular detection and quantification methodology for bacteria and the importance of the assessment of its efficiency is evident, as is the case in fungal diagnostics [START_REF] Fredricks | Comparison of six DNA extraction methods for recovery of fungal DNA as assessed by quantitative PCR[END_REF]. Following the results of this study, FTA ® Elute paper would be our method of choice for use on-site with samples taken from solid surfaces, based on processing time per sample, easiness of use, DNA extraction efficiency, broad-range species effectiveness, chemical toxicity and transport and storage advantages.

  The probes were labelled with carboxyfluorescein (FAM) on the 5"-end and a 4-([4-(dimethylamino)phenyl]azo)benzoic acid (DABCYL) quencher on the 3"-end. All qrtPCR reactions were performed in a total volume of 30 µl consisting of 3 µl of target DNA and 27 µl of amplification mixture containing PCR Reaction Buffer (Smart™ Kit No ROX, Eurogentec S. A.), primers and probe and Molecular Biology Water (AccuGENE ® , Lonza Group Ltd, Basel, Switzerland). The Smart™ Kit PCR reagents contained HotGoldStar DNA polymerase and Uracil-N-Glycosylase to prevent carry-over contamination. The end concentration of each primer was 150 nmol l -1

  DNA extractions were performed in quadruplicate. The efficiency of DNA extraction was determined by the measured amount of bacterial DNA in each sample as compared to the estimated amount of bacterial DNA before DNA extraction as a reference (100%). The reference was estimated by microscopic cell counting and assuming a chromosomal weight calculated from the chromosome length and GC content of approximately 3.0 fg for S. aureus and 5.7 fg for E. coli, respectively. Within each DNA extraction method, efficiencies of extraction amongst different cell amounts were compared by Kruskal-Wallis analysis. Friedman analysis with additional Bonferroni-protected contrasts was performed to compare DNA extraction methods amongst each species. Differences were considered statistically significant when P values were less than 0.05 (two-sided), resulting in 0.05/(number of contrasts performed) after Bonferroni correction. SPSS Inc. [http://www.spss.com/]) and Microsoft ® Office Excel 2003 (Microsoft Corporation [http://office.microsoft.com/]) software, respectively.

  ATCC11755 and S. aureus subsp. aureus ATCC 25923 compared to the other two methods. As shown by the results, the measured amount of DNA in some cases exceeded that of the reference which was estimated by the "gold standard" of microscopic cell counting, resulting in percentages above 100 percent. An underestimation of the amount of chromosomal DNA by microscopic cell counting can easily occur due to factors such as cell clumping, cell division or cell lysis.

  reactions produced a positive result.

Fig 1 Fig 2 Fig 3 Online Resources 1 and 2

 1232 Fig 1 DNA levels extracted by three methods with varying amounts of cells. Median DNA levels as determined by qrtPCR resulting from quadruplicate extractions (n=4) of different amounts of cells of (A) E. coli ATCC 11775 T and (B) S. aureus subsp. aureus ATCC 25923. Extractions were performed by three different DNA extraction methods: FTA ® Elute, the Reischl et al. method and QIAamp ® DNA Mini Kit. Error bars indicate minimum and maximum values. Extrapolated quantification limits of the methods are represented by dotted lines: ---, FTA ® Elute, ─ ─ ─, Reischl et al. and QIAamp ® DNA Mini Kit.

  Reischl et al. method, 110 minutes for FTA ® Elute and 5 hours 30 minutes for the QIAamp ® DNA Mini Kit. These processing times did not include preparation of the solutions. The sample input volumes were 90 µl for the Reischl et al. method, 40 µl for the FTA The qrtPCR quantification limit was determined at 30 fg of the standard for E. coli and at 10 fg of the standard for S. aureus, respectively. Sampling in the toilet area was positive for E. coli with a mean of 4.50±0.33 log fg DNA (~3.18 × 10 4 fg), as can be expected since the potential pathogen E. coli is a common inhabitant of the human gut. This amount of DNA would correspond with ~5.58 × 10 3 E. coli cells per sample. In contrast, the mean S. aureus and E. coli DNA content from a location between wall panels was below the cut-off level.

	Figs. 1A and 1B show the median DNA levels measured per cell amount after extraction, their
	corresponding reference and the extrapolated limits of quantification for each DNA extraction
	method.
	The median efficiencies of DNA extraction of different cell amounts calculated for each DNA
	extraction method are presented in Table 1. Within each method, no significant differences were
	observed by Kruskal-Wallis analysis amongst efficiencies of DNA extraction of different cell
	amounts. Figs. 2A and 2B give an overview of the median efficiency of DNA extraction of the 5.3
	× 10 5 , 5.3 × 10	4 and 5.3 × 10	3 cell amounts for each method. P values of P≤0.017 were
	considered statistically significant for the performed three contrasts after Bonferroni correction.
	The experiments show that for E. coli ATCC 11775	T the FTA	® Elute paper had the highest
	median DNA extraction efficiency of 76.9 percent, followed by the Reischl et al. method with 43.7
	percent. The QIAamp
				09 × 10	7 cells
	0.01ml -1 (cumulating variation coefficient (CVC)=10.52) and 9.51 × 10 6 cells 0.01ml -1 (CVC= protocol was significantly lower with 9.0 percent as compared to the QIAamp ® DNA Mini Kit
	9.76), with a mean of 1.02 × 10 9 cells ml -1 . In addition, the concentration of the S. aureus cell (P=0.001) and FTA
	suspension was determined in duplicate at 3.47 × 10 7 cells 0.01ml -1 (CVC=9.43) and 3.23 × 10 7
	cells 0.01ml -1 (CVC=10.72), with a mean of 3.35 × 10 9 cells ml -1 .
	QrtPCR standard curves with E and R
	Subsequently, when assuming a Ct equal to the mean Ct (+ 1 s. d.) of the qrtPCR quantification
	limit, extrapolated limits of quantification per extraction could be determined for E. coli to be 552 The applicability of the FTA ® Elute method was examined by extracting DNA with this method
	fg DNA for FTA ® Elute and 137 fg DNA for the Reischl et al. method and QIAamp from swab samples taken from the interior of the ISS. For S. aureus, a potential pathogen ® DNA Mini Kit.
	For S. aureus extrapolated limits of quantification per extraction were determined at 961 fg DNA commonly found on the human skin, a location associated with human contact was positive with a
	for FTA mean of 4.50±0.21 log fg DNA (~3.16 × 10

® Elute paper and 180 µl for the QIAamp ® DNA Mini Kit. Output volumes for the samples were 100 µl for the Reischl et al. method, approximately 13.4 aliquots of 30 µl for the FTA ® Elute paper and 100 µl for the QIAamp ® DNA Mini Kit. The concentration of the E. coli cell suspension was determined in duplicate at 1.2 values that were determined for E. coli and S. aureus are presented in Online Resources 1 and 2. ® Elute and 238 fg DNA for the Reischl et al. method and QIAamp ® DNA Mini Kit. ® DNA Mini Kit had the significantly lowest median DNA extraction efficiency of 7.7 percent as compared to FTA ® Elute (P=0.001) and the Reischl et al. method (P=0.004). For S. aureus subsp. aureus ATCC 25923, FTA ® Elute paper and QIAamp ® DNA Mini Kit performed with similar efficiencies of 108.9 and 97.7 percent, respectively. The efficiency of the Reischl et al. ® Elute paper (P=0.001). The level of PCR inhibition of the DNA extraction methods on the E. coli qrtPCR assay is presented in Fig. 3. The results show that compared to added water to 1 ng of the qrtPCR standard DNA, both the added extraction controls of the Reischl et al. method and QIAamp ® DNA Mini Kit caused a shift towards a higher Ct, indicating an inhibitory effect, in contrast to the extraction control of the FTA ® Elute method. No inhibitory effects were noticeable on the S. aureus qrtPCR assay (data not shown). 4 fg). Calculated from the chromosomal weight, this amount of DNA would correspond with ~1.05 × 10 4 S. aureus cells per sample.

Table 1

 1 Efficiencies of DNA extraction of different amounts of cells of E. coli ATCC 11775 T and S. aureus subsp. aureus ATCC 25923 by three different methods: FTA ® Elute, the Reischl et al. method and QIAamp ® DNA Mini Kit.

	Number of cells		5.3 ×	5.3 ×	5.3 ×	5.3 ×	5.3 ×
			10 5	10 4	10 3	10 2	10 1
			Median % (n=4)
	E. coli ATCC 11775 T	FTA ® Elute	40.5	84.8	96.3	BC d	BC d
		Reischl et al.	48.9	59.6	24.5	BC b	ND
		QIAamp ® DNA Mini	4.6	32.3	13.5	BC b	BC c
		Kit
	S. aureus subsp. aureus ATCC	FTA ® Elute	112.6	94.9	119.4	BC b	BC c
	25923
		Reischl et al.	10.2	8.8	8.5	BC b	BC a
		QIAamp ® DNA Mini	69.4	117.0	97.7	149.9	BC
		Kit

a ND, not detected; BC, Below Cut-off level, i.e. not all of the reactions were positive and

Acknowledgements

We especially like to thank A. M. Borries, S. van der Schaaf and G. C. Raangs for their technical contributions to this project. We thank W. J. Sluiter for statistical advice and G. W. Welling for critically reading the manuscript. None of the authors had any degree of commercial or potential dual interest with the manufacturers mentioned in this study. This project was supported by the European Space Agency (MAP Project Number AO-LS-99-MAP-LSS-018 "Biofilms").