P Hogström
email: per.hogstrom@chalmers.se

J W Ringsberg

E Johnson

An experimental and numerical study of the effects of length scale and strain state on the necking and fracture behaviours in sheet metals

Keywords: Digital Image Correlation (DIC), Finite element analysis, Forming limit test, Length scale dependence, Sheet metal failure, Tensile test

Within sheet metal forming, crashworthiness analysis in the automotive industry and ship research on collision and grounding, modelling of the material failure/fracture, including the behaviour at large plastic deformations, is critical for accurate failure predictions. In order to validate existing failure models used in finite element (FE) simulations in terms of dependence on length scale and strain state, tests recorded with the optical strain measuring system ARAMIS have been conducted. With this system, the stress-strain behaviour of uniaxial tensile tests was examined locally, and from this information true stress-strain relations were calculated on different length scales across the necking region. Forming limit tests were conducted to study the multiaxial failure behaviour of the material in terms of necking and fracture. The failure criteria that were verified against the tests were chosen among those available in the FE software Abaqus and the Bressan-Williams-Hill (BWH) criterion proposed by Alsos et al. 2008. The experimental and numerical results from the tensile tests confirmed that Barba's relation is valid for handling stress-strain dependence on the length scale used for strain evaluation after necking. Also, the evolution of damage in the FE simulations was related to the processes ultimately leading to initiation and propagation of a macroscopic crack in the final phase of the tensile tests. Furthermore, numerical simulations using the BWH criterion for prediction of instability at the necking point showed good agreement with the forming limit test results. The effect of pre-straining in the forming limit tests and the FE simulations of them is discussed.

Introduction

Sheet metal forming processes, crashworthiness in the automotive industry, and ship collision and grounding are examples of areas in which numerical models play an important role in process development, in the design of full-scale tests/prototypes, and as a substitution for fullscale tests. The finite element (FE) method is an established numerical tool suitable for these purposes. However, there is still a need to continuously study, improve and validate the influence of many parameters in the numerical simulations and models due to ever increasing demands for more accurate predictions of, for example, material failure or for mimicking the responses recorded in tests that have been carried out. Often, both the accuracy and reliability of numerical models and what they aim to represent are discussed, together with whether or not their applicability is general or limited to specific conditions or types of analyses, see Alsos et al. [START_REF] Alsos | Analytical and numerical analysis of sheet metal instability using a stress based criterion[END_REF]. This investigation focuses on the analysis of structures made of thin steel sheets considering their structural resistance to fracture as a result of large plastic deformations caused by, as one example, a collision between two cars or two ships. The objective was to analyze and compare models for material failure using an explicit FE software. Results from FE simulations are compared with experimental results from uniaxial tensile tests and forming limit (FL) tests.

Consequently, the influence of the element size of the mesh, the length scale dependence on the failure limit and damage evolution models can be studied in detail. Additional topics that are addressed are the influence of multiaxial strain state on failure (see Alsos et al. [START_REF] Alsos | Analytical and numerical analysis of sheet metal instability using a stress based criterion[END_REF]) and the applicability and reliability of the criteria studied in numerical investigations of ship-ship collision events. The most frequently used criteria for predicting material failure are based on effective

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3 plastic strain. Such criteria have gained popularity due to their simple and effective formulation and have proven to give results with satisfying accuracy by Karlsson et al. [START_REF] Karlsson | Experimental and numerical investigation of bulb impact with a ship side-shell structure[END_REF], among others.

However, these criteria neglect the influence of strain state, and recently, criteria which take this into account have been proposed and applied, see e.g. Hong et al. [START_REF] Hong | Damage assessment and impact resistant design of FPSOs with respect to supply vessel collisions[END_REF]. An example of such a criterion, which was compared with the experiments, is the Bressan-Williams-Hill (BWH) criterion proposed by Alsos et al. [START_REF] Alsos | Analytical and numerical analysis of sheet metal instability using a stress based criterion[END_REF] in 2008.

In the experiments, the ARAMIS optical strain measuring system [START_REF]ARAMIS[END_REF] was used, which enabled accurate monitoring of displacements on the specimen surfaces. A brief description of this system is given in Section 2. When a material reaches the limit of its capacity to carry further loading, deformations localize into necking and become highly dependent on the length over which the strain evaluation is performed -the length scale. In FE simulations, this length scale dependence must be taken into account when using elements of different sizes in the mesh, as discussed by Lehmann and Peschmann [START_REF] Lehmann | Energy absorption by the steel structure of ships in the event of collisions[END_REF] and Hong et al. [START_REF] Hong | Damage assessment and impact resistant design of FPSOs with respect to supply vessel collisions[END_REF]. This issue, as well as the applicability of Barba's relation, are addressed in Section 3. Furthermore, the strain axiality also has an impact on the necking and fracture behaviour of the material which should be accounted for in FE simulations. These effects can be investigated with information from FL tests as discussed by Ramaekers [6], Ragab [START_REF] Ragab | Prediction of fracture limit curves in sheet metals using a void growth and coalescence model[END_REF], Zhao et al. [START_REF] Zhao | A theoretical and experimental investigation of limit strains in sheet metal forming[END_REF], and Stoughton and Zhu [START_REF] Stoughton | Review of theoretical models of the strain-based FLD and their relevance to the stress-based FLD[END_REF]. This type of test is described in Section 4. In Section 5, an analytical solution of the BWH criterion [START_REF] Alsos | Analytical and numerical analysis of sheet metal instability using a stress based criterion[END_REF] is presented and applied on the forming limit test results. The capacity of some failure criteria available in the FE software Abaqus/Explicit is investigated in Section 6. This section also presents the results from the FE simulations of the tests. Note that throughout the current investigation stresses and strains are always the true stresses and strains unless otherwise stated.

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The ARAMIS system and the material tested

The optical strain measuring system ARAMIS [START_REF]ARAMIS[END_REF] was used in the tests to make precise measurements of the displacements on the surface of the specimens tested. The measuring system consists of two digital cameras connected to a computer with an image recognizing software matching the images from the cameras. Based on these images, the ARAMIS system can identify points on the specimen surface and divide it into facets (pixels). The distortion of these facets is monitored throughout a test in three-dimensional space. Prior to the tests, a thin coating with a stochastic pattern, which the ARAMIS system uses to identify facets and follow their displacements, was added to the specimen's surface. The random pattern on the surface of the specimen was used to follow the deformation as well as fracture behaviour of the specimen material. The resulting facet displacement shows, for instance, regions of localized strains. The accuracy in the results of displacements given from the ARAMIS system depends greatly on the size of the test specimen and the test setup. The manufacturer claims the accuracy of displacement measurement to be in the interval 0.01 -0.1 pixels. In the test setup of the current study, the size of the pixels was 0.05 mm, giving an accuracy of at least 0.005 mm. The cameras used in the present study allow for a constant sampling frequency of 1 Hz, and the computer used has the capacity to store up to 500 images during one test.

Three materials were tested in the current investigation: the NVA mild steel [START_REF]Rules for classification of ships/high speed, light craft and naval surface craft[END_REF], the Domex 355 high strength steel [11], and the NV5083 aluminium material [START_REF]Rules for classification of ships/high speed, light craft and naval surface craft[END_REF]. The NVA material is a material that is commonly used in shipbuilding today, while the frequency of use of Domex

Tensile tests

The tensile tests were conducted in accordance with the DNV rules [START_REF]Rules for classification of ships/high speed, light craft and naval surface craft[END_REF] but with the exception that the restriction of loading speed in the elastic region was disregarded. This was done because the elastic behaviour of the material was not a focus of the current study.

Instead, the speed was set to give as high resolution in time as possible throughout the test given the sampling frequency and ability to store output data of the ARAMIS system, see Section 2. According to Dowling [START_REF] Dowling | Mechanical Behaviour of Materials 3rd edition[END_REF], mild steel materials similar to the NVA grade used in the current investigation may have an engineering fracture strain of up to 40%. The gauge length of the test specimen was 78 mm [START_REF]Rules for classification of ships/high speed, light craft and naval surface craft[END_REF], which, with a speed of 5 mm/min in the test, allowed for an engineering strain of up to 50% at the end of the test. Fig. 1 shows the engineering stress-strain curves for the different materials from the tests. Note that there was some margin between the largest engineering strain in the test and the maximum limit of 50% strain.

Fig. 1: Typical engineering stress-strain curves for the tested materials, NVA, Domex 355 and NV5083 Fig. 2 shows the test setup for the tensile test. In order to have full control of movements out of the plane, two reference plates were introduced in the experimental setup: one was fixed to the machine foundation, and the other plate followed the moving crosshead of the tensile test machine. The specimens were manufactured according to the DNV rules [START_REF] Zhao | A theoretical and experimental investigation of limit strains in sheet metal forming[END_REF], oriented with the loading direction aligned with the rolling direction of the sheet panel they were

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6 manufactured from. Thus, the specimens were slightly curved prior to the test. Since the ARAMIS system only measures displacements on one surface of the test specimen, the system cannot distinguish between straightening of the specimen and its cross-sectional area contraction.

However, by studying the reference plate attached to the moving crosshead of the tensile test machine, a distinction between these effects could be made: while the test specimen was undergoing elastic deformation, a slight rotation of the reference plate could be seen. Around the point of plastic yielding this rotation ceased, indicating that the test specimen had been straightened by the machine during the elastic deformation. By assuming that the area contraction of the test rod was small at this point, the area calculated there could be used as the initial area in the stress calculations. The "Aramis" stress in Fig. 3 was calculated using the force recorded by the load cell in the test machine divided by the actual area of the crosssection where fracture eventually occurred. This area was calculated using the displacement information recorded on the specimen. In Figs. 3a, 3c and 3e, the stress-strain relationships obtained from the tensile tests carried out on the NVA, the Domex 355 and the NV5083 materials are presented. The data obtained from the ARAMIS recordings allow for an analysis of the displacements over length scales defined by the engineer. As a result, the "Aramis" strain of the test rod can be measured using virtual extensometers (VE), which represent various length scales. A VE is defined as the distance between two points along the length of the test rod positioned at an equal distance from the point of fracture. A long VE corresponds to a strain value measured over the entire length of the test rod (see the bold line in Figs. 3a, 3c and 3e), while a small value of the VE corresponds to more local strain behaviour (see the dotted lines in Figs. 3a, The curves fitted to the fracture points in Figs. 3b, 3d and 3f are a relation with one asymptote in the global fracture strain for the whole test rod and one asymptote where the length of the virtual extensometer approaches zero. This is known as Barba's relation and in the current investigation a formulation proposed by Yamada et al. [START_REF] Yamada | Numerical study on the effect on buffer bow structure in ship-ship collision[END_REF] was used. It expresses the fracture strain as:

        + = VE f L Wt c e n ε ε ln ( 1
)
where e is the mathematical constant defined as the base of the natural logarithm, W is the original width of the specimen (25 mm), t is its original thickness (4 mm), ε n is the necking strain (0.22), and L VE is the VE length over which the fracture strain is measured. The Barba parameter, c, was calibrated to obtain a curve that matches the results obtained from the experiments. A comparison of the c-values for the materials shows that the higher the ultimate elongation of a material, the larger the value of c, as seen in Figs. 3 and4.

Forming limit tests

Forming limit tests were conducted to study the multiaxial strain behaviour of the material in terms of necking and fracture. The tests were limited to the NVA steel grade, which was selected because it is the most commonly used material in shipbuilding of the three materials studied in the current investigation. The tests were carried out in accordance with the ISO 12004-2 standard [12], and the ARAMIS system [START_REF]ARAMIS[END_REF] was used to monitor the surface displacements of the test specimens. Six different geometries were tested, shown in Fig. 5a, corresponding to six data points in the forming limit diagram (FLD). Three samples of each geometry were tested. The specimens were manufactured from the same sheet panel as the tensile test specimens with the orientation of the rolling direction perpendicular to the major principal strain direction of the test section of the specimen. They were clamped by bolts in the fixture and deformed quasi-statically normal to their plane using a half spherical punch with a diameter of 100 mm, see the test setup in Fig. 5b. A lubrication layer consisting of three layers of silicon grease and two sheets of reinforced Teflon was added between the test specimen and the punch in order to reduce the influence of friction. According to standard

[12], the velocity of the punch should be 1.5 ± 0.5 mm/s. However, a velocity of 0.1 mm/s was used instead because it gives a higher resolution in time of the results as determined by the sampling frequency of the cameras of the ARAMIS system (see Section 2). A low

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9 velocity is also beneficial to reduce the influence from strain rate effects (note that strain rate effects were not incorporated in the current study). The identification of the location of necking and fracture in a specimen and when it occurs during the test was determined in accordance with the ISO 12004-2 standard [12], and Fig. 6 shows an example of results in a diagram that was used for this purpose. The procedure behind the determination of fracture is presented first, followed by necking.

In the time step preceding fracture of a test specimen, the major principal strain, ε 1 , in five sections perpendicular to the crack was calculated using the information from facet displacements recorded by the ARAMIS system. One section was put in the middle of the crack with two sections on each side, approximately 2 mm apart. The dots in Fig. 6 show an example of typical results in one section of a test geometry for the major principal strain. The ISO 12004-2 standard [12] prescribes that the strain values close to the crack should be excluded in the evaluation, i.e. the strain values that form the peak in Fig. 6 should be omitted in the assessment. Instead, ten strain values on each side of the crack, represented here by the squares in Fig. 6, should be used to fit an inverse second-degree-polynomial, shown by the solid line in Fig. 6. Once this polynomial has been obtained, the major principal strain of the section under consideration is defined as the peak value of the polynomial, represented by the

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10 triangle in Fig. 6. As a result of using this method, one major principal strain value for each of the five sections is obtained. The mean value of them is the major principal strain for fracture of the test specimen. The minor principal strain, ε 2 , is evaluated similarly at the time step which defines the major principal strain, ε 1 , at fracture.

Fig. 6:

Example of evaluation procedure of the forming limit test results in accordance with ISO 12004-2 [START_REF]Rules for classification of ships/high speed, light craft and naval surface craft[END_REF] to obtain the major principal strain, ε 1 , in one of the five evaluation sections of a test specimen. The position 0 along the test section (x-axis) corresponds to the location of the crack.

Necking can be defined as a localization of displacements (see Hill [START_REF] Hill | On discontinuous plastic states, with special reference to localized necking in thin sheets[END_REF]). Utilising this definition together with the measurement data from the ARAMIS system, a methodology is proposed as to how to calculate the principal strains at necking. It partly follows the methodology prescribed by the ISO 12004-2 standard [START_REF]Rules for classification of ships/high speed, light craft and naval surface craft[END_REF], but some additional steps had to be developed and incorporated for clarity and to ensure quality of the results. The specimen under consideration was divided into five sections, perpendicular to the crack, for evaluation in accordance with the ISO 12004-2 standard [START_REF]Rules for classification of ships/high speed, light craft and naval surface craft[END_REF]. In every section, the major and minor principal strains were calculated in the facets using the measurement data from the ARAMIS system. These strains were also calculated as a function of time; see Fig. 7 for an example of the major principal strain in one section of a test geometry. The diagram presented in Fig. 7 was used to identify where localization takes place in the specimen, depicted by the dashed line. Following the same procedure as described in the previous paragraph for fracture principal strains, ten points on each side of the dashed line were chosen for evaluation of the characteristics of the principal strains, such as the square symbols in Fig. 6. The time history

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11 of the evolution of the principal strains at these points was of main interest in the evaluation of necking principal strains.

For the given geometries and loading conditions of the present investigation, the major principal strain was considered to be the driving strain parameter in the evolution of localization of displacements. Fig. 8a shows an example of the time history of the major principal strain for the twenty points (cf. the squares in Fig. 6) used in the assessment in one section of a test specimen. The solid line is the standard deviation between these data as a function of time. It may serve as an indicator for when localization (i.e. necking) has already occurred by its exponential increase. In order to define necking in time, the acceleration of the principal strain (second-order derivative of principal strain) was chosen as a measure. In Fig. 8b, the major principal strain acceleration curves corresponding to the curves in diagram Fig. 8a are presented. The solid line is the standard deviation of the major principal strain acceleration curves and can be described by Eq. ( 2):

( )

2 / 1 2 1 1 2 , 1 2 2 , 1 2 )
( 1 ) ( 1 1                 - - = ∑ ∑ = = q i q j j i dt t d q dt t d q t Q ε ε (2)
where q is the number of points in the assessment (here, q = 20), and ε(t) 1,i and ε(t) 1,j are the major principal strains in these points. As necking can be considered to occur almost instantaneously (or very rapidly), the point in time when the standard deviation increases significantly was used as a definition of necking, represented by the dashed line in Fig. 8b. At this point in time, a procedure similar to the definition of principal strains at fracture according to standard [12] was used: the ten points (facets) on each side of the dashed line in Fig. 7 (cf. the crack and squares in Fig. 6) were used to fit an inverse second-degree- The summary of the results from the forming limit tests are presented in the principal strain space in Fig. 9. Here, the mean values of each specimen are presented, and the error bars denote the standard deviations in test results between test specimens for the test geometries 1 to 6, respectively. It should be noted that even though the evaluation procedure [12] is designed to reduce the scatter [START_REF]Rules for classification of ships/high speed, light craft and naval surface craft[END_REF], there is larger scatter in results from the measurements for fracture in the ε 1 direction in contrast to the ε 2 direction. When establishing a strain based FLD, proportional strain path has to be assumed [6,[START_REF] Zhao | A theoretical and experimental investigation of limit strains in sheet metal forming[END_REF][START_REF] Stoughton | Review of theoretical models of the strain-based FLD and their relevance to the stress-based FLD[END_REF]. However, since the forming limit curve (FLC) is not only dependent on the material but also [START_REF] Zhao | A theoretical and experimental investigation of limit strains in sheet metal forming[END_REF] performed FL tests on sheet material with different induced pre-straining using a similar test setup as the present study, showing that the pre-straining has a significant impact on the strain path. The strain paths in their experiments on rolled material show the same tendency as the FL tests, an example of which can be seen in Fig. 10 in which the strain paths of the five evaluation sections of one specimen of geometry 4 are shown. Strain in the minor principal direction comes towards the end of the test, mainly after the point of necking, giving the curvature in strain path. It may also be seen for the other geometries in Fig. 9 where the points representing necking are gathered closely together for geometries 2-6. Geometry 1 does not show this behaviour since it is fixed around its circumference. Zhao et al. [START_REF] Zhao | A theoretical and experimental investigation of limit strains in sheet metal forming[END_REF] showed that strain paths differ between materials with different induced pre-strain, which indicates that the curvature in strain path lies within the pre-straining rather than the test setup in the present case. Stoughton and Zhu [START_REF] Stoughton | Review of theoretical models of the strain-based FLD and their relevance to the stress-based FLD[END_REF] argue in favour of using a stress based FLD in order to remedy the strain path dependency of the strain based FLD. However, going from a strain based to a stress based FLD requires some calculations based on assumptions. Thus, the comparison between experimental and numerical results are made in the strain based FLD in which the experimental results can be directly determined from the displacements measured by the ARAMIS system.

Description of a forming limit curve using the BWH criterion

Alsos et al. [START_REF] Alsos | Analytical and numerical analysis of sheet metal instability using a stress based criterion[END_REF] have proposed a criterion for the onset of instability (necking) in sheet metal, which is referred to as the BWH criterion. It searches for local instability and applies only to ( )

( )          + + ≤         + + + + + + = otherwise ) 2 /( 1 3 / 2 3 2 0 if , 1 ) 1 ( 3 2 1 5 . 0 1 3 2 2 1 2 1 2 1 β β ε β β β β ε β β β σ n c n c K K (3) here, 1 2 / ε ε β & & =
, K is the material strength coefficient, n is the strain hardening exponent [START_REF] Dowling | Mechanical Behaviour of Materials 3rd edition[END_REF] and ε 1c is the local necking in a uniaxial strain state. The ε 1c parameter is the major principal strain at the onset of necking where the FLC crosses the major principal strain axis.

Examining the points of necking in the FLD in Fig. 9 reveals that a curve fitted to these points crosses the major principal axis at ε 1 ≈ 0.27. In the current study, the strain hardening exponent, n, was obtained by a curve fit of the power stress-strain relationship to the tensile test results to n = 0.22. With this value and ε 1c = 0.27, the BWH curve on the strain-based form in Fig. 11 fits the test results well. When going from a stress to a strain based formulation of the BWH criterion, some assumptions have to be made, such as the plane stress state along with a formulation of effective stresses (von Mises equivalent stresses and strains have been used here). In order to express a relationship between principal strains, proportional strain paths of each geometry point in the FLD, expressed as constant

1 2 1 2 / ε ε ε ε β = = & &
throughout the test, are assumed

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15 [START_REF] Alsos | Analytical and numerical analysis of sheet metal instability using a stress based criterion[END_REF]. The investigation and discussion of strain paths presented in Fig. 10 indicates that this assumption may not be valid in the initial path preceding necking. Nevertheless, because experimental results are in the strain space and the deviation from proportional strain path in Fig. 10 is small compared to similar investigations (e.g. Zhao et al. [START_REF] Zhao | A theoretical and experimental investigation of limit strains in sheet metal forming[END_REF]), the assumption of constant β was used.

Finite element simulations and results

The finite element model of the test specimens used in the tensile and forming limit tests consisted of shell elements with reduced integration (S4R in Abaqus/Explicit) and five section points through their thickness. Generally, shell elements that are thick in relation to their side lengths give poor results in bending; this type of element has a plane stress formulation and thus they are unable to resolve stress gradients in their thickness direction. In the uniaxial tensile tests, no bending is present. In the forming limit tests, membrane and bending stresses are present, but the membrane stresses are regarded as being dominant, making the use of shell elements feasible. In addition, results in the elements are taken in the through-thickness mid section point, i.e. in the neutral axis, and thus bending stresses are disregarded.

The material modelled was the NVA grade material represented by an isotropic hardening model with piecewise linear isotropic hardening characteristics for the plastic behaviour. A Young's modulus of 210 GPa and a yield stress of 310 MPa were used, as shown in Fig. 2a.

In the simulation of the forming limit tests, zero friction was assumed, and the contact conditions between the punch and the specimen were modelled using the "general contact condition" in Abaqus/Explicit. Only half of the test specimens were modelled because of symmetry.

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16

For the modelling of different physical phenomena leading to failure of a material, Abaqus/Explicit offers several models that handle initiation and evolution of damage. For damage initiation (DI) in ductile metals [16], either the ductile criterion, a phenomenological model for the nucleation, growth and coalescence of voids, or the shear criterion, which models shear band localization, may be used. In the current investigation, the ductile criterion was used to model the damage initiation in the FE simulation of the tensile tests using the necking strain from the tests, ε n = 0.22, as the plastic strain at the onset of damage. In addition to these models, several criteria for simulating instability of sheet metal are available, such as the "FLD criterion" that was used in the simulation of the forming limit tests [16]. In Abaqus/Explicit, criteria for sheet steel can only be used with elements that have a plane stress formulation, i.e. shell, beam or truss elements.

After the damage initiation, a damage evolution model describes the degradation of the material up to the point of fracture. In Abaqus/Explicit, the evolution is defined either through the displacement at fracture, u f , or the energy dissipated during the failure process, G f . The former alternative was used in the current study. The displacement at fracture is defined as

u f = L×ε f
where L is a characteristic element length, and ε f is the plastic strain at fracture taking into account the influence of the length scale, cf. Eq. 1. In the post-necking region, the element size of the mesh has a great influence on the solution (see the discussion in Section 3). Consequently, this dependency has to be accounted for when the damage evolution parameter is defined. In Abaqus/Explicit, damage evolution may be defined as linear, bilinear/piecewise linear, or following an exponential behaviour.

Finite element simulations of the tensile tests

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17

The FE simulations of the tensile tests were conducted in order to study the damage evolution law in terms of mesh dependency (element size or length scale) and choice of damage evolution function. Several element sizes (between 2 and 8 mm) were compared to investigate its influence on the solution and results. Up to the point of damage initiation, no dependence from the element size could be seen between different FE models and simulations. However, after the initiation of damage, the influence from the element size became significant. This could be compensated for by using Barba's law by adjustment of the u f -parameter according to the characteristic element length in the FE model, as seen in the previous section.

The stress-strain curve in Fig. 12a that represents the experiments was plotted up to the point where a macroscopic crack started to propagate in the test specimen. This can be compared with Fig. 3a where two distinct phases of the true stress-strain relationship can be seen after necking: first a slow increase of the stress that then accelerates towards the final fracture when the macroscopic crack occurs and propagates. The two curves "linear" and "bilinear" damage evolution (DE) in Fig. 12a were obtained by FE simulations, and Fig. 12b gives their definitions. The FE simulations were interrupted at a point that had a similar definition as the experimental point of interruption of a test, i.e. when the gradient of the stress-strain relation increased rapidly and fracture occurred. The displacement at fracture, u f , used in the FE simulations was calculated using ε f according to Barba's law, see Eq. ( 1). In Fig. 12a, the results obtained by FE simulations using the model with linear damage evolution show a deviation in stress-strain behaviour at the point of necking in comparison with the experimentally obtained results. It may also be noted that it cannot mimic the damage process and seems to underestimate the energy released during the damage process. This could be remedied if the damage variable, D, is allowed to evolve according to a bilinear relationship, the results of which are shown in Fig. 12b. However, when defining such a relationship, the D-u f relation has to be calibrated so that it results in the same amount of dissipated energy in order to reach zero stiffness at the same strain as the linear damage formulation, as can be seen in the illustration of the damage evolution in Fig. 12b. Using the bilinear damage evolution relationship, a calibrated value of the x-parameter to 0.2 resulted in excellent agreement with the experimentally obtained stress-strain curves, shown in Fig. 12a.

A slower development of damage in the early phase and a more rapid evolution towards final fracture shows a closer resemblance to the two phases of the experimental observations leading to macroscopic crack initiation and propagation, as discussed above. This behaviour of acceleration towards final fracture was also observed in the FE simulation using the linear DE, but, in this particular case, it had to be enhanced using a bilinear law to match the test results.

Fig. 13 shows a comparison in results between a tensile test and an FE simulation of it. Fig. 13a shows the major principal strain in the facets on the specimen's surface, which was calculated using the data recorded by the ARAMIS measurement system. The corresponding result from an FE simulation is shown in Fig. 13b, in which the bilinear damage evolution relationship was used with x = 0.2. The results are presented at the time, T, which is 95% of the total time to fracture, T f . There is very good agreement in results between the experiment

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and the FE simulation with respect to magnitude of the major principal strain and the contours of its distribution.

Fig. 13: Major principal strain results for a tensile test presented at T = 0.95T f :

(a) results from an experiment using the ARAMIS system and (b) results from an FE simulation using Abaqus/Explicit.

Finite element simulations of forming limit tests

In the FE simulations of the forming limit tests all six geometries in Fig. 4a were assessed. To define damage initiation, which represented the necking in the experiments, the FLD criterion in Abaqus/Explicit was used with tabular values of ε 1 and ε 2 taken from the BWH curve presented in Fig. 11 as input. Degradation due to evolution of damage was represented in the FE model using the bilinear damage evolution law used also in the FE simulation of the tensile test (x = 0.2) (see Fig. 12b). The results from the FE simulations were evaluated similarly to the evaluation of tests; Section 4 presents details regarding this method.

The results presented in Fig. 14 show that the trends of both the necking and fracture are captured by the FE simulated values; however, some discrepancies are present. The points representing necking for the test geometries 2-6 are collected around the major principal strain axis, while the corresponding simulated points are more separated in the ε 1 -ε 2 space. A similar trend can be seen with the points representing fracture. One reason for this effect may be that, as was discussed in Section 4, the material of the specimens was pre-strained (and anisotropic), which was not represented in the FE material model. Fig. 15 shows a comparison in results of the major principal strain in a test specimen of geometry 2 and the FE simulation of the same geometry. The results are presented at a time, T, which is 95% of the total time to fracture, T f . There is very good agreement in results both with respect to magnitude of the major principal strain and the contours of its distribution.

Fig. 15:

Major principal strain results for a FLD test on geometry 2 at T = 0.95T f :

(a) results from an experiment using the ARAMIS system and (b) results from an FE simulation using Abaqus/Explicit.

Discussion

This investigation presents an experimental and numerical investigation on necking and fracture behaviour of sheet metals on a small scale, i.e. on specimens. Depending on the type of application and objective of the FE analysis, one may use either necking or fracture as the definition of failure in the analysis. Also, the sizes of the elements forming the model have to be adjusted in combination with the selected interruption criterion for element removal or damage evolution. In large structures like ships, large sizes of the elements that form the mesh are recommended in order to save computational efforts. Then, necking may be a sufficient criterion for failure and element removal, and it has been successfully used by, among others, Alsos [17,[START_REF] Alsos | On the resistance to penetration of stiffened plates, Part II: Numerical analysis[END_REF], who utilised the BWH criterion for this purpose. Marciniak et al. [START_REF] Marciniak | Mechanics of Sheet Metal Forming[END_REF] also consider necking to be the point of failure, since the load carrying capacity of a structure consisting of sheet material is lost after the point of necking. numerous assumptions concerning the mechanisms involved in necking of sheet metal, the procedure for adapting the criterion to experimental values [START_REF] Alsos | Analytical and numerical analysis of sheet metal instability using a stress based criterion[END_REF] gives a good correlation with the results from the forming limit tests. Furthermore, using this criterion within the FE analysis of the forming limit tests captures the trend of the experimental values for necking.

The discrepancy between experimental and simulated values may be explained by the prestraining and anisotropy of the tested material, an effect which was not accounted for in the material description of the FE models in the present study.

In future work, the model of failure established within the current investigation will be incorporated into large scale FE simulation of ship-to-ship collisions, an example of which is shown in Fig. 16. In Abaqus/Explicit, the FLDCRIT variable is used as a measure to present the initial accumulation of damage of the material between the yielding point, FLDCRIT = 0, and the point of necking, FLDCRIT = 1 if the FLD-criterion is used. The SDEG variable shows the evolution of damage in an element between the necking point, SDEG = 0, and fracture, SDEG = 1. When SDEG = 1, the elements are removed from the mesh to visualize if, for example, a crack/hole has occurred in the side-shell of the ship, as seen in Fig. 16a. Fig. 16b shows the result from the same analysis and time step as in Fig. 16a but for the SDEG variable. It should be noted that in this type of simulation, many different sizes of elements will be used in order to resolve the ship structure appropriately. In that way, the failure behaviour dependence on element size established in experiments and FE analysis of the tensile test, like in Figs. 2 and12, should be used. On the other hand, the elements will most likely be of such a size that their failure strain is close to the global strain of the tensile test, cf. the asymptotic relations in Figs. 3b, 3d and 3f, in which case this global failure strain will be used. However, in failure analysis of other applications where smaller elements are used,

M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS

22 this dependence will have a large impact on the solution and should be compensated for.

Also, it is beneficial to have a law of damage evolution when conducting FE analysis using Abaqus/Explicit, since this mitigates numerical instabilities that may occur if large stiffness gradients are present in the solution. Furthermore, the geometry of a ship structure consists of large plates (cf. forming limit test geometry 1 in Fig. 5a) and narrow stiffeners (cf. forming limit test geometries 2-6 in Fig. 4a) in which very different stress states will be present. In FE analysis, this will be compensated for by using the FLD criterion for damage initiation in Abaqus/Explicit with input data from the analytical solution of the BWH instability relation fitted to experimental data.

M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS

23

A method to identify the point of instability in sheet metal through this definition of necking is proposed for forming limit tests. Concerning the post-necking behaviour of the material, this was modelled in FE simulations using a bilinear law for damage evolution found through observations on tensile tests. The same evolution law was applied to the FE simulations of the forming limit tests which then correlate well with the trends in the experiments.

The method presented in Alsos et al. [START_REF] Alsos | Analytical and numerical analysis of sheet metal instability using a stress based criterion[END_REF] to apply the BWH criterion to experimental values of necking at the forming limit tests showed good agreement between test values and those predicted by the criterion. In terms of using the FLD criterion in Abaqus/Explicit, with input from the BWH criterion adapted to the experimental points of necking and the damage evolution found in the tensile tests, the results from the simulations capture the trend of the experimental necking and fracture FLCs well. In the comparison of the results from the tests and the FE simulations of the forming limit test geometries 2 to 6 (see Sections 4 and 6.2), the results from the FE simulations are more separated in the ε 1 -ε 2 space in contrast to the test results. This difference can be explained by the pre-strain and anisotropy that is caused by the manufacturing process of the material tested-an effect which was not incorporated in the description of the material characteristics in the FE model.

  355 and NV5083 materials have increased lately as a consequence of the demands on weight reduction and lighter ship structures. The thickness of the specimens was 4 mm, and they were manufactured from hot-rolled steel plates and cold-rolled aluminium plates. The M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS 5 experimental setup fulfilled the requirements in the ISO 12004-2 standard [12] and the DNV rules [10].

Fig. 2 :

 2 Fig. 2: Test setup for the tensile test.

  . Traditionally, necking is identified as the point in the engineering stress-strain diagram where the stress reaches its maximum. See the vertical line in Figs.3a, 3c and 3efor their corresponding true strain values. Another definition which is used here is the point in the true stress-strain diagram where the different VEs diverge, i.e. when the strains localize. Note that the two definitions of necking show good resemblance.

Fig. 3 :

 3 Fig. 3: Results from the tensile tests where the stress is calculated based on the actual area of

Fig. 4 :

 4 Fig. 4: Three tensile test rods after testing which are made of the NVA, the Domex355 and

Fig. 5 :

 5 Fig. 5: (a) The six test geometries: each of them corresponds to one strain state in the forming

  M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS12 polynomial, which, finally, after evaluation of all of the five sections of the specimen, gives the principal strain values for necking.

Fig. 7 :

 7 Fig. 7: Example of a presentation of the evolution in time of the major principal strain in one

Fig. 8 :

 8 Fig. 8: (a) Curves for the major principal strain history in the points of the evaluation window

Fig. 9 :

 9 Fig. 9: Results from forming limit tests of the six geometries (based on three test samples per

  and geometry, a straight strain path is rarely the case in experiments, as concluded by Ramaekers [6]. Zhao et al.

Fig. 10 :

 10 Fig. 10: Strain path during the forming limit tests for five sections of one test specimen

  strains, i.e. bending is disregarded in this criterion. It is described in Eq. (3) on a stress-based form:

Fig. 11 :

 11 Fig. 11: Results (necking) from forming limit tests together with the analytical solution of the

Fig. 12 :

 12 Fig. 12: (a) Simulated tensile tests using a linear and a piecewise linear law for damage

Fig. 14 :

 14 Fig. 14: Results presented in principal strain space from the experiments and FE simulations

  BWH instability criterion used in the current investigation is based on

Fig. 16 :

 16 Fig. 16: Example of results from an FE simulation of a ship-to-ship collision event. Part of

Fig. 1 :Fig. 2 :Fig. 3 :Fig. 4 :

 1234 Fig. 1: Typical engineering stress-strain curves for the tested materials, NVA, Domex 355 and NV5083

Fig. 5 :

 5 Fig. 5: (a)The six test geometries: each of them corresponds to one strain state in the forming limit diagram. Geometry 1 is the circular plate (upper left) and geometry 6 is the narrowest of the geometries (lower right). (b) Test setup: the punch goes into the hold in which the specimen is clamped, the Teflon lubrication sheets can be seen between them. Also, the displacement gauge can be seen fixed on top of the punch.

Fig. 6 :

 6 Fig. 6:Example of evaluation procedure of the forming limit test results in accordance with ISO 12004-2[START_REF]Rules for classification of ships/high speed, light craft and naval surface craft[END_REF] to obtain the major principal strain, ε 1 , in one of the five evaluation sections of a test specimen. The position 0 along the test section (x-axis) corresponds to the location of the crack.

Fig. 7 :

 7 Fig. 7: Example of a presentation of the evolution in time of the major principal strain in one section of a test geometry. The dashed line indicates where in the specimen localization is expected to occur.

Fig. 8 :

 8 Fig. 8: (a) Curves for the major principal strain history in the points of the evaluation window (see squares in Fig. 5) and their standard deviation enhanced by a factor of 10. (b) Major principal strain acceleration of the curves in (a) and their standard deviation enhanced by a factor of 10. The dashed line indicates necking.

Fig. 9 :Fig. 10 :Fig. 11 :

 91011 Fig. 9: Results from forming limit tests of the six geometries (based five sections on three test samples for each geometry): mean values and standard deviations (denoted by the error bars) in the principal strain space (ε 1 and ε 2 directions), for necking and fracture.

Fig. 12 :

 12 Fig. 12: (a) Simulated tensile tests using a linear and a piecewise linear law for damage evolution (DE). Note that the solid line representing the experiments and the dash-dotted line representing the FE simulations using the bilinear damage evolution coincide. (b) Illustration of linear and piecewise linear DE relationships during the process of damage evolution.

Fig. 13 :Fig. 14 :Fig. 15 :Fig. 16 :

 13141516 Fig. 13: Major principal strain results for a tensile test presented at T = 0.95T f : (a) results from an experiment using the ARAMIS system and (b) results from an FE simulation using Abaqus/Explicit.

Acknowledgements

This investigation has been carried out as part of the research project HASARD (Holistic assessment of ship survivability and risk after damage). The authors acknowledge the Swedish Governmental Agency of Innovation Systems (VINNOVA) and the Swedish Competence Centre in Maritime Education and Research, LIGHTHOUSE (www.lighthouse.nu), for financing the project. The authors are also grateful to the staff at the division of Building Technology and Mechanics at SP Technical Research Institute of Sweden in Borås, Sweden for providing expertise and support in conducting the experiments.


G Christner

H Ackermann

Rohde

Dr Corina Ilchmann

Angelika Speicher

Nils Henning Zaiß

Ackermann Grit

Holger Rohde

Martin Christner

Grit

2 3

Keywords: Clostridium difficile, PCR ribotype, resistance, epidemiology, binary toxin

Comparison of resistance against erythromycin and moxifloxacin, presence of binary toxin gene and PCR

ribotypes in isolates from 1990 and 2008

Ackermann, and 1 Holger Rohde 5 6 1 Institut für Medizinische Mikrobiologie, Virologie und Hygiene, Universitätsklinikum 7 Hamburg-Eppendorf, Hamburg, Germany, 2 Robert Koch Institute, Burgstrasse 37, 38855 8 Wernigerode, Germany, 3 (MIC 50 : 256 µg/ml vs. 1.5 µg/ml; MIC 90 : 256 µg/ml vs. 32 µg/ml) and moxifloxacin (MIC 50 : 9 32 µg/ml vs. 0.75 µg/ml; MIC 90 : 32 µg/ml vs. 1 µg/ml). 40 / 73 (54.8 %) of the isolates from 10 2008 were resistant against erythromycin and moxifloxacin, an isolated resistance against 11 erythromycin was detected in 4 / 73 (5.5 %) isolates and against moxifloxacin in 2 / 73 (2.7 12 %) isolates (Table 1). Overall, compared with C difficile strains from 1990 the proportion of 13 erythromycin and moxifloxacin susceptible strains was significantly lower in C.difficile 14 isolates from 2008 (20/23, 87 % vs. 27/73, 37 %, p<0.001). Importantly, 37 / 44 (84.1 %) 15 isolates belonging to PCR ribotype 001, but only 2 / 29 (6.9 %) strains belonging to PCR 16 ribotypes other then 001 were resistant against erythromycin and moxifloxacin. All cdtB-17 positive isolates from 2008 were susceptible against both agents. 18 Epidemiological evidence suggest that on one hand occurrence and spread of variant C. 19 difficile PCR ribotype 027 contributed to the ever increasing number of CDI cases in regions 20 with epidemic outbreakes [1]. However, spread of this strain is clearly not the exclusive 21 reason for the changing epidemiology and clinical presentation of CDI in general. The low 22 prevalence of ribotype 027 found in our study is in line with a study investigating the 23 distribution of PCR ribotypes in a European collection of 411 C. difficile isolates, assigning 24 only 6,2% of all isolates to PCR ribotype 027 [5]. In southern Germany CDI was 25 predominantly caused by C. difficile belonging to ribotype 001 [6]. In line with this study we here document the importance of PCR ribotype 001 in our hospital, and it is tempting to 1 speculate that ribotype 001 is of special importance in Germany [START_REF] Arvand | Clostridium 1 difficile ribotypes 001, 017, and 027 are associated with lethal C. difficile infection in 2 Hesse[END_REF]. 2 Interestingly, we were able to demonstrate presence of cdtB-positive C. difficile ribotype 027 3 isolates already in a collection of 23 strains from 1990, demonstrating that ancestors of the 4 current epidemic clone were present in virulent C. difficile populations almost 20 years ago. 5

Detailed molecular comparison of these early strains with current C. difficile 027 isolates 6 could provide new insights into the evolution of this particular strain, which is a prerequisite 7 for understanding the molecular basis for its epidemic potential. 8

It is well accepted that antibiotic treatment is a major risk factor for C. difficile gut 9 colonization, infection and the subsequent development of CDI [1]. We here document a 10 significantly increased prevalence of erythromycin and moxifloxacin resistant C. difficile 11 isolates between 1990 and 2008. It should be emphasized that it cannot be excluded that this 12 finding results from differences between patients groups sampled in 1990 and 2008, rather 13 than differences between C. difficile populations. In addition, the results could be biased by 14 2. The authors try to demonstrate "epidemiologic changes", however, this is not possible due to limitations of the study design. Therefore, the present characterisation of these ad hoc collected C. difficile isolates is certainly of interest for the local situation but to very less extent to the scientific community.

We have removed the respective sentence.

General comments

At the end of the manuscript it should clearly stated. The results of the present study are limited due to several reasons...... In order to fit with the requirements for a brief report we saw no possibility to add the statement. However; the limitation referee 1 is referring to are mentioned in the text (page 5, line 12 ff:" It should be emphasized that it cannot be excluded that this finding results from differences between patients groups sampled in 1990 and 2008, rather than differences between C. difficile populations. In addition, the results could be biased by the different length of periods used for sampling.")

Abstract line 6 please change "The present study" in " The present single center retrospective analysis"

The sentence was changed accordingly.

line 14 please substitute:

No isolates belonging to PCR ribotype 027 were identified "in our cohort". Sentence was changed.

line 17 and other parts of the manuscript inclucing page 7 line 8:

The term "epidemiology" should not be used throughout the complete manuscript in this retrospective analysis with unselected samples. The term was, if referring to our observations, deleted.

4. page 6 lines 3-4: "variant C. difficile PCR ribotype 027 contributed to the ever increasing number of cases" please substitute "in regions with epidemic outbreakes"

The sentence was changed.

5. page 6 line 14: "we here document the importance of PCR ribotype 001"please substitute "when analyzing the more recent isolates"

The sentence was modified accordingly.

6. Please add a comment into the discussion concerning differenced betwee the historical 027 strains detected in 1990 and the new epidemic outbreak strains.

We have no detailed knowledge about these differences. However, we highlight the possibility to use isolates from 1990 for comparative analysis with recent outbreak strains (page 5, lien 3 ff: almost 20 years ago. Detailed molecular comparison of these early strains with current C. difficile 027 isolates could provide new insights into the evolution of this particular strain, which is a prerequisite for understanding the molecular basis for its epidemic potential."

A direct comparison of antibiotic susceptibility and binary toxin between the 1990 and 2008 strains with the same ribotypes may be of interest

We agree with the referee. However, we think that due to the small numbers of certain ribotypes this analysis in our collection is not meaningful.

8. Page 6 line 22: "The rare occurrence of cdtB in our and independent C. difficile collections [8,11] shows that the ability to produce binary toxin is of minor importance for increase of CDI rates," The study design is not appropriate to give a statement about the clinical importance of binary toxins. Only descriptive analysis is allowed.

To cope with this point of criticism we have changed the sentence, removing the statement related to our findings.

Reviewer 2

The term fluorquinolon was corrected throughout the text.

Page 5, line 1. The authors should consider inserting the 87% proportion as stated in the abstract, to describe the number of isolates susceptibles to both clones. Otherwise, the 87% proportion cannot be found in the main text of the manuscript.

We followed the suggestion.page 3, line 24 ff: "……while 20 / 23 (87 %) were susceptible against moxifloxacin and erythromycin (Table 1).") Page 5, line 18 For the same reason as above, 87% should be mentioned again in this sentence, so that it is clear which are the two proportions that are being compared. A suggestion: "Overall, compared with C difficile strains from 1990 the proportion of erythromycin and moxifloxacin susceptible strains was significantly lower in C.difficile isolates from 2008 (20/23, 87% vs 27/73, 37%, p<0.001).

We followed the suggestion.

  the different length of periods used for sampling. However, as especially fluoroquinolones 15 have more recently been identified as an important risk factor for the development of CDI [8], 16 it is reasonable to speculate that a host environment significantly exposed against 17 fluoroquinolones favours the expansion of resistant C. difficile clones [9]. Indeed, several 18 other studies lend support to this idea [10], demonstrating that significant proportions of 19 clinical C. difficile isolates from different PCR ribotypes are resistant against 20 fluoroquinolones. Strikingly, in our collection erythromycin and fluoroquinolone resistance 21 was associated with isolates belonging to PCR ribotype 001, suggesting that the over all 22 increase of erythromycin and moxifloxacin resistant C. difficile isolates is in part related to 23 the spread of this ribotype. The multi resistant phenotype of C. difficile PCR ribotype 001 24 could, in addition to its ability to sporulate more frequently [11], explain the particular 25 epidemic potential of this strain.

here to download Manuscript: 100429 Revised2.doc Click here to view linked References prevalence

  MVZ für Laboratoriumsdiagnostik und Medizinische Mikrobiologie 9 of resistant C. difficile strains. This single centre retrospective analysis 5 characterized resistance against erythromycin and moxifloxacin, presence of binary toxin 6 gene and ribotypes in 73 C. difficile isolates from 2008 in comparison with 23 isolates from 7 1990. In 1990 five different PCR ribotypes including 027 were identified. Resistance against 8 erythromycin was detected in 3 / 23 (13 %), while 20 / 23 (87 %) of all isolates were 9 susceptible to both, erythromycin and moxifloxacin. In contrast, in 2008 a significantly 10 increased prevalence of resistant C. difficile strains was observed, with 40 / 73 (54.8 %) 11 isolates being resistant against both antibiotics. Resistant C. difficile strains were mainly 12 assigned to PCR ribotype 001. No isolates belonging to PCR ribotype 027 were identified. 13 Our data provide evidence that increase of resistant C. difficile strains belonging to PCR 14 ribotype 001 rather than the spread of C. difficile PCR ribotype 027 contribute to the changing 15 Table 1). Both cdtB-positive isolates belonging to ribotype 25 027 were susceptible to erythromycin and moxifloxacin. In 2008 a total of 16 different PCR ribotypes were determined. Most isolates (44 / 73; 60.3 %) were assigned to ribotype 001, 25 1

	*Manuscript
	Dr. Ackermann GmbH, Riebeckstraße 65, 04317 Leipzig, Germany Corresponding author: PD Dr. Holger Rohde, Institut für Medizinische Mikrobiologie, Virologie und Hygiene, Martinistraße 52, 20246 Hamburg, Germany; tel.: 0049 40 7410 52143; fax: 0049 40 7410 53250; e-mail: rohde@uke.uni-hamburg.de Clostridium difficile has long been considered a significant causative agent of nosocomially Click epidemiology of CDI. 10 11 12 13 14 15 16 17 18 19 20 1 16 17 Key words: Clostridium difficile, PCR ribotype, resistance, binary toxin 18 19 acquired diarrhoea [1]. Since 2002 increasing numbers of infections, outbreaks and severe 2 cases of Clostridium difficile-infections (CDI) have been reported. In part, these infections 3 were attributed to variant C. difficile strain belonging to PCR ribotype 027 [1]. This strain 4 produces binary toxin CDT, encoded by cdtA and cdtB, and has a partial deletion of the 5 regulatory tcdC gene, resulting in toxin A and B hyperproduction [2]. In addition, 6 independent PCR ribotypes (e.g. 001, 053, 078 and 106) can cause outbreaks and severe 7 courses of C. difficile infections [3], indicating that currently epidemiology of virulent C. 8 difficile strains is subject to complex changes that cannot solely be attributed to the spread of 9 one particular virulent C. difficile strain. 10 We here analyzed C. difficile isolates from patients hospitalized at the University Hospital 11 Centre Hamburg-Eppendorf in 1990 (n=23) and 2008 (n=73) with clinical suspicion of CDI. 12 Isolates from 1990 were randomly collected over the whole year. These stool samples were 13 not tested for the presence of toxin A or B. No information about antibiotic therapies was 14 available. C. difficile isolates from 2008 were consecutively collected during a three month 15 period from patients hospitalized in different departments. 39 / 73 (53.5 %) of the stool 16 samples were toxin positive (TechLab Quik Check, Blacksburg, USA). At the time of sample 17 collection, 47 / 73 64.3 %) patients received antibiotics (11 / 73 [15.1 %], no antibiotics. 15 / 18 73 [20.6 %], no information). 19 Using a published method [4] C. difficile isolates from 1990 were assigned to 5 different PCR 20 ribotypes (012, n=3; 014, n=8; 027, n=2; 042, n=9; 053, n=1). PCR detected cdtB in 2 / 23 21 (8.7 %) isolates, both belonging to PCR ribotype 027. Susceptibility testing using the Etest 22 (Biomerieux, Marcy l´Etoile, France) revealed that only 3 / 23 (13 %) C. difficile isolates 23 were resistant against erythromycin, while 20 / 23 (87 %) were susceptible against 24 2 020, n=1; 023, n=1; 032, n=1; 042, n=1; 045, n=1, 046; n=1; 066, n=1; 078, n=1; 081, n=2; 3 087, n=1). Four strains were not typeable, PCR ribotype 027 was not detected. cdtB was 4 found in only 3 / 73 (4.1 %) isolates belonging to PCR ribotypes 023, 066, and 078, 5 respectively. Statistical analysis revealed that cdtB prevalences in 1990 and 2008 did not 6 differ significantly (p=0.590, Fisher´s exact test). However, compared to C. difficile isolates 7 moxifloxacin and erythromycin (isolates belonged to sporadic ribotypes (003, n=3; 010, n=1; 012, n=1; 014, n=8; 015, n=1; from 1990, C. difficile isolates from 2008 exhibited increased MICs against erythromycin 8

Table 1 : Distribution of erythromycin and moxifloxacin MIC values in C. difficile isolates from 1990 and 2008 Antimicrobial agent (year) No. of strains for which the antimicrobial agent MIC (µg/ml) was* Geometric mean (µg/ml)

 1

		0,125	0,25	0,5	0,75	1 1,5 2 4	8	16	32	256
	ERY (1990)		2	1	3	2	8	4			1	2	24.75
	ERY (2008)	2		2	3	7 15 1			43	151.27
	MX (1990)			6	9	8					0.77
	MX (2008)			14	11	7					41	18.28

* Breakpoints used to differentiate susceptible and resistant isolates were defined for ERY at ≥ 4 µg/ml (according to DIN) and for MX at ≥8 µg/ml (according to CLSI)

Table Reviewer 1

 Reviewer 1. This is a very short five page long paper with very few data. Only one table with original data is given (table 1) whereas the MIC of table 2 can be directly calculated from the original data in table 1.Table2was removed, geometric means were included in table 1.

Response to Reviewers' Comments Click here to download Authors' Response to Reviewers' Comments: 100429 Reply.doc

Interestingly, we were able to demonstrate presence of cdtB-positive C. difficile ribotype 027 isolates already in a collection of 23 strains from 1990, demonstrating that ancestors of the current epidemic clone were present in virulent C. difficile populations *Authors'

Acknowledgements 1

The authors thank Wolfgang Witte for critically reading the paper. There was no financial 2 support for the study. All authors state that there are no potential conflicts of interest. 3 4