Concrete is the most commonly used structural material, without which modern construction could not function. It is a material with a high potential to adapt to specific operating conditions. The use of this potential is made by its material modification. The aim of the performed investigations was the assessment of rational application possibilities of fly ashes from thermally conversed municipal sewage sludge as an alternative concrete admixture. A concrete mix was designed, based on the Portland cement CEM I 42.5R and containing various quantity of ash, amounting to 0–25% of cement mass. The samples were conditioned and heated in a furnace at the temperature of 300 °C, 500 °C, and 700 °C. Physical and chemical properties of the ashes as well as utility properties of the concrete, i.e., density, compressive strength after 28, 56, and 90 days of maturation, frost resistance, and compressive strength in high temperature were determined. The tests were performed at cubic samples with 10 cm edge. The replacement of a determined cement quantity by the fly ashes enables obtaining a concrete composite having good strength parameters. The concrete modified by the fly ashes constituting 20% of the cement mass achieved its average compressive strength after 28 days of maturation equal to 50.12 MPa, after 56 days 50.61 MPa and after 90 days 50.80 MPa. The temperature growth weakens the composite structure. The obtained results confirm the possibility of waste recycling in the form of fly ashes as a cement substitute in concrete manufacturing. The current legislation imposes increasingly complex requirements on building materials. It is aimed at safety, durability, and ecological friendliness of buildings. In the document Model Code 2010 ,4. Numer2 resulting from the decarbonization of raw material [2, imposed by the European Union [Portland cement manufacturing technology is a process with a lot of disadvantages, comprising the consumption of a big quantity of natural resources, very high energy consumption, and significant environmental pollution, including high emission of COmaterial ,8,9,10. an Union , prompt Nowadays, calcareous and siliceous fly ashes originating from the combustion of hard and brown coal are widely applied in concrete manufacturing technology . The cruPer the requirements for quality control procedures, the chemical and physical properties for fly ash used as a type II additive in the concrete production are specified in PN-EN 450-1:2012 norm ,14,15. I2 [Sewage sludge produced in sewage treatment plants are a big environmental problem. They are often utilized with the use of incineration, which produces a fly ash. The main goal of the conducted experimental work is to examine the possibility of using ashes from sewage sludge in the concrete production ,31,32,332 ,36,37. I2  of dry m2 , and the2 , the res2 ,42,43. T2 ,45 consi2 , 20% of 2 . Some re2 ,49,50. IDestructive changes in the concrete composites containing the Portland cement are evoked by high temperatures that occur during fire. In previous studies , it has This paper is a continuation of the authors previous works, and aside from concrete strength features with various content of fly ashes (FA) from thermally converted municipal sewage sludge, it presents their frost and high temperature resistance. The aim of experimental research was to evaluate the effect of temperature on frost resistance strength parameters of concretes produced with the use of fly ash from the thermal treatment of sewage sludge. The article also presents tests results and analyses of fly ash from sewage sludge properties.In aim to perform investigations, an ordinary concrete mix of class C20/25 and consistency S3 was designed with the use of a calculation and experimental method based on the Bukowski’s three equations method. The main components of concrete mix are: Portland cement CEM I 42.5R from Cement Ożarów S.A. , sand aggregate of 0–2 mm fraction, gravel aggregate 2–16 mm fraction, fly ashes from thermally conversed sewage sludge from Cracow, and water. In all samples, the same granulometric composition of the fine aggregate (0–2 mm), selected with use of the sieve analysis, and the same composition was assumed of coarse aggregate selected with subsequent iterations  Table 1Table 1. With the aim to perform a comparative analysis of properties of the ordinary concrete and concretes containing the fly ashes from thermally converted municipal sewage sludge, mixes were prepared with various contents of admixture , as shown in The concrete mix consistency was checked with use of concrete slump test , the buc,cub150, fc,cub100 denotes the compressive strength observed on cubic samples with the edge length equal to 15 and 10 cm, respectively.The compressive strength tests were performed according to PN-EN 12390-3:2019 directives, after 28, 56 and 90 days of maturation . The tesp = 0.95. With the aim to assess the expanded  uncertainty, the sclerometric unscaled method was used [The frost resistance tests were performed with use of a direct method according to the procedure described in the standard PN-88/B-06265 in the Toropol cold chamber . In the was used .The thermal resistance of the composite against high temperature was investigated in a special furnace PK 1100/5 from Termolab S.C. . The samples were held in the temperature 300, 500 and 700 °C. The furnace had electrically supplied heating sections. The rig is equipped in the dedicated program ThermoPro  which enables programming of heating process. The temperature distribution in the tested element was monitored with use of the thermocouples NiCr-Ni, fulfilling the standard requirements .Before the start of the heating process, all samples had been dried out to a constant mass at the temperature of 105 °C ± 5 °C. The signal from the thermocouples was recorded by a computer equipped with the dedicated software. The tests were performed according to a standard curve temperature vs. time. With the aim to determine time of holding in individual temperatures, specifically prepared samples with borings for measuring thermocouples were subjected to pilot tests. The temperature distribution was monitored by 4 thermocouples . The borAiming to recognize fly ashes tested, the following characteristics were determined: (1) the chemical composition using the energy dispersive X-ray fluorescence (XRF) method; (2) the mineral composition using X-ray phase analysis (XRD); (3) the grain size distribution using a laser diffraction; (4) the morphology and chemical composition using scanning electron microscope technique; and (5) the pozzolanic activity according to the standards ,67 and rStatistical methods are useful for interpretation and modeling of large data sets. In this study, variance analysis (ANOVA) was used. ANOVA analysis is used to determine the difference of more than two independent variables. SPSS program  was used to analyze the data.2 32.21%), aluminum dioxide (Al2O3 6.25%), and iron oxide (Fe2O3 19.25%) in the sewage sludge fly ashes did not satisfy the requirements contained in the standard PN-EN 450-1+A1:2012 and concerning siliceous fly ashes (min. 65%) [3, the fineness according to the standard PN-EN 451-2:2017-06 [2-17.8), aluminum oxide (Al2O3-11.1), and iron oxide (Fe2O3-6.5) in these ashes was lower and amounted to only 35.4.Analysis results of fly ashes from municipal sewage sludge composition are presented in in. 65%) . The grain. 65%)  was equa:2017-06  46.2%, t:2017-06  and PN-E:2017-06  0.5 mm. It was observed that the fly ash samples were dominated by irregular grains with variable size with highly porous, strongly developed surface as well as loose and rough structure. Spherical and cuboidal forms were very rare.The SEM-EDS chemical microanalysis proved the domination of the silicon, aluminum, phosphorus, and iron grains . Such co2 is affected by their chemical and mineral composition. The performed investigations of fly ashes from thermally conversed sewage sludge pozzolanic activity allowed to assess the application possibilities of this admixture in concrete modification. According to the standard ASTM C379-65T [2 and Al2O3. The pozzolanic activity, established in the performed investigations, was equal to 20.49%. The total content of the aforementioned oxides over 20% indicates a pozzolanic character [The fly ashes demonstrate the pozzolanic activity, i.e., they react with calcium hydroxide in its presence (it can originate from hydrolysis of klinker cement siliceous phases), creating products having hydraulic and binding properties ,73,74,75C379-65T , the pozharacter ,77. Accoharacter , the flyharacter .2O/Al2O3). The so-called desirable pozzolanic potential, introduced by these authors, classifies fly ashes into three classes depending on this oxide ratio: 1st class-K2O/Al2O3 ·10 ≥ 1, 2nd class-0.5 < K2O/Al2O3 ·10 < 1, 3rd class-K2O/Al2O3 ·10 < 0.5. For the investigated fly ash, a result 0.34 was obtained which categorizes the fly ash as little reactive-class 3.According to Hubbard and Dhir , the fly3 (FA20%). The smallest air amount was observed in the concrete mix of reference concrete OC, equal to 1.7%, while the highest, equal to 2.8% was in the mix FA20% -2371 kg/mix FA20% .Results of s of average compressive strength measurement of concrete samples with various content of the fly ashes from thermally conversed sewage sludge is presented in cm within the range of 93.4% (FA2.5%) – 98.7% (FA20%). Replacing cement with ash from sewage sludge in an amount greater than 10% causes an increase in strength in relation to the reference concrete without the addition  achieved 90.4% of the final strength (after 90 courses of curing). Concretes modified with fly ash from thermal treatment of sewage sludge reached values faddition . The res2O5, CaO, SiO2, and Al2O3 compounds has a fundamental influence on the concrete properties. An increase in the share of these oxides in the composition of fly ash leads to a decrease in compressive strength. On the other hand, the increase in the sum of silicon content, aluminum, and iron oxide in the ash composition increases the compressive strength.The concentration of PThe frost resistance test consists in determining the decrease in compressive strength of a sample frozen at a temperature of ± 20 °C in relation to a non-frozen sample. The reduction in compressive strength should not exceed 20%. According to the PN-88/B-06250  standardConcrete modified with fly ash from sewage sludge up to 15% is frost-resistant concrete F150. The lowest loss in strength was observed for the sample with 2.5% of cement replaced by the ashes (FA2.5%), whereas the highest-for the FA20% samples (with 20% ash content). The values amounted 2.69% and 22.69%, respectively. The average loss in mass was slight in all cases and oscillated between 0.251% (FA15%) and 1.037% (FA5%). The compressive strength of reference concrete samples ranged from 44.62 MPa (for FA2.5%) to 57.25 MPa (for FA20%). After 150 cycles of freezing and thawing at a temperature of ± 20 °C, it was observed that the highest compressive strength equal to 44.56 MPa was achieved by concrete in which 20% of cement was exchanged for fly ash. On the other hand, the lowest strength of 42.26 MPa was achieved by concrete without the addition of CO.A temperature increase weakens a material structure. Cracks and scratches were visible on the sample surface. A real example temperature distribution presented in The thermocouples distributed in the pilot sample allowed modeling of a standardized fire development. A heating process was programmed by setting heating time, temperature and time sections number of the process which had to be realized. The point where temperatures equalize shows that the sample is evenly heated in its volume. In the self-cooling phase temperature increase of external layers is visible what is confirmed in investigations .After holding in the temperature 300 °C, it was observed an increase of the average compressive strength, related to the strength determined for the room temperature, and it was equal to 9.0 MPa for the OC concrete and 2.9 MPa for the FA5% concrete. For the remaining concretes, the compressive strength fell for the FA10% by 3.6MPa, for the FA15% by 8.1 MPa, and for the FA20% by 10.4MPa.Fire temperatures exceeding 500 °C evoke a decrease in the compressive strength by ca. 11% for the FA5%, 24% for the FA10%, 38% for the FA15%, and 30% for the FA20%. The concretes in the temperature 700 °C reduced their strength in the range between 31% (OC) and 61% (FA15%) related to the samples made of the reference concrete (not heated). A lower content of the fly ashes from thermal conversion of sewage sludge in concretes positively affects the compressive strength of the samples subjected to the action of fire.p < 0.05) (p < 0.95). There is a statistical difference in all temperature measurements for FA10%, FA15%, and FA20%.In this study, one-way ANOVA analysis was performed to determine the differences between compressive strength, temperature, and fly ash amount. According to ANOVA analysis results, the parameters of OC, FA5%, FA10%, FA15%, and FA20% among different temperature  are statistically significant different ( < 0.05) . A furthp < 0.05) (p < 0.95). For the samples tested, after 150 freeze-thaw cycles, there is a statistical difference between OC and FA12.5%, FA17.5%, and FA20%, except for FA2.5%, FA5%, FA7.5%, FA10%, and FA15% (p < 0.95).ANOVA analysis was also performed to determine the differences between compressive strength of control samples (samples that were not subjected to cyclic freeze-thaw) and after 150 freeze-thaw cycles. According to ANOVA analysis results, the values of compressive strength are statistically significant different ( < 0.05) . For theFrom an ecological point of view, the re-use of wastes from the thermal conversion of sewage sludge in the concrete manufacturing technology brings economic advantages. The investigations proved that the fly ash can perfectly substitute cement as a hydraulic mineral binder obtained from mineral raw materials.There is a possibility of using waste in the form of fly ash from the thermal treatment of sewage sludge as an additive  for the concrete production. Fly ash in the amount of up to 20% does not deteriorate the compressive strength and frost resistance.The highest average compressive strength, equal to 50.12 MPa, 50.61 MPa, and 50.80 MPa, after 28, 56, and 90 days of maturation, respectively, was obtained by concrete FA20%, in which 20% of cement was replaced with fly ash from thermal treatment of sewage sludge. The lowest average compressive strength of 39.74 MPa, 41.64 MPa, and 42.52 MPa after 28, 56, and 90 days of maturing was achieved by concrete FA2.5%. All concretes reached the designed (assumed) concrete class C20/25.The chemical and physical composition of the fly ashes from sewage sludge is different than that of fly ashes from the combustion of hard coal and does not fulfill the standard PN-EN 450-1:2012 requirements. The highest percentage share in the fly ash samples corresponded to silicon, calcium, phosphorus, and aluminum oxides. However, there are no regulations concerning the physical and chemical properties of ashes from sewage sludge incineration that would limit the possibilities of their use in concrete technology.The pozzolana activity of the fly ash does not meet the applicable requirements of the PN-EN 450-1:2012 standard after 28 (≥75%) and 90 days (≥85%) of maturation. Fly ash from sewage sludge reaches the required values after a longer maturation period, which allows it to be classified as an active mineral additive.Concrete samples containing in their composition fly ash from incineration of sewage sludge were characterized by a comparable average compressive strength to the comparative concrete without the addition. If the fly ash content does not exceed 20% of the cement mass, the ashes can be applied as a cement substitute.The investigations showed that the concretes containing 2.5–15% of the cement mass are frost-resistant. Concrete made on the basis of fly ash from sewage sludge was characterized by the best values of compressive strength after 150 cycles of freezing and thawing. A slight strength increase was observed as the content of fly ashes from municipal sewage sludge rose within the range 2.5–15% of the cement mass.After heating the concrete samples to the temperature of 300 °C, a compressive strength increase was observed by 3 MPa for the FA5% concrete and by 9 MPa for the OC concrete. The temperature increase to 700 °C caused the compressive strength to decrease in the range between 31% (OC) and 61% (FA15%).During annealing at the temperatures 300–700 °C, the samples did not experience any thermal chipping, which is undoubtedly beneficial due to its destructive and dangerous course.The temperature influence caused the structure damage of tested concrete surface, visible scratches appeared on the outer surfaces. This phenomenon is common due to Portland cement use.After the analysis of the results and observations made during the performed experimental investigations, it can been stated that:The concrete composite modified with the waste product from the thermal conversion of sewage sludge is characterized by satisfying usable parameters, which was confirmed by the performed experiment.It is difficult to find in sources any information concerning the influence of the fly ashes from the thermal conversion of sewage sludge on the strength parameters and frost resistance of concrete composites. Information concerning the resistance of these concretes against high temperature is also limited. The facts presented in this paper constitute a basis for further investigations within this framework. Due to regulations and lack of and standards concerning the application of fly ashes from the thermal conversion of sewage sludge, any further interpretation of detailed results requires supplementary investigations.


Generating random aggregate models (RAMs) plays a key role in the mesoscopic modelling of concrete-like composite materials. The arbitrary geometry, wide gradation, and high volume ratio of aggregates pose a great challenge for fast and efficient numerical construction of concrete meso-structures. This paper presents a simple strategy for generating RAMs of concrete based on Laguerre tessellation, which mainly consists of three steps: tessellation, geometric smoothing, and scaling. The computer-assisted design (CAD) file of RAMs obtained by the proposed approach can be directly adopted for the construction of random numerical concrete samples. Combined with the image-based octree meshing algorithm, the scaled boundary finite element method (SBFEM) was adopted for an automatic stress analysis of mass concrete samples, and a parametric study was conducted to investigate the meso-structural effects on concrete elasticity properties. The modelling results successfully reproduced the increasing trend of concrete elastic modulus with the grading of coarse aggregates in literature test data and demonstrate the effectiveness of the proposed strategy. Concrete is widely accepted as a composite material, and the main constituents at the meso-scale are commonly categorized into aggregate, mortar matrix, and interfacial transition zone. The internal heterogeneous structure contributes most to its complex behavior under a variety of loading conditions. Amongst the three-phase constituents, coarse aggregates normally occupy a large volume portion of a concrete mix, and their properties, such as size, shape, gradation, arrangement, and strength can influence the physical and mechanical properties of the final concrete batch to a great extent. Much attention has been given in literature to the effects of meso-structures on concrete properties, using theoretical, experimental, or numerical approaches. As an alternative to standard experimental techniques, numerical testing considering the meso-level particle structure  has becoFurthermore, mass concrete containing maximum-size aggregates up to 150 mm are widely used in massive concrete structures, such as dam projects and offshore structures. In the dam engineering industry in China, dam concrete containing combined aggregate gradations of 5–20 mm, 20–40 mm, and 40–80 mm is classified as three-graded concrete, and termed as four-graded or fully graded concrete when another grade of aggregate of 80–150 mm is mixed together ,4, the mIn the 1980s, Wittmann et al. , first uFor continuum meso-scale numerical investigation into concrete materials, finite element discretization is another challenging task following the generation of numerical aggregates. Owing to the randomness and irregularity of the polyhedron aggregates, convex or concave, constructing hexahedral mesh elements for representing concrete meso-structures, which are normally preferred in most circumstances, still remains a great challenge. When an accurate characterization of the outer surfaces of aggregate solids is demanded, the aligned meshing approach based on tetrahedral elements become a viable choice . On the Following the second category of approach for generating RAMs mentioned above, this paper presents a simple and efficient method to generate three-dimensional RAMs. Specifically, Laguerre tessellation is considered as the initial bounding boxes for aggregates, and the actual grading requirements can be satisfied by a proposed grading adjustment strategy. Furthermore, based on the CAD file of RAMs, the SBFEM program combined with the octree algorithm is used to mesh irregular-shaped aggregates and the matrix component automatically, and the factors affecting elastic constants of large aggregate concrete are discussed from a mesoscopic point of view.Borrowing the ideas from the layering disposition method  and the The construction of Voronoi tessellation is commonly used to represent the polycrystalline structure and can effectively divide the specimen space into a collection of polyhedrons without overlaps and gaps by putting random seeds inside and generating lines and faces around each seed. These entities are defined as influence zones of a particular set of seeds. Recently, the development of Laguerre tessellation, a generalized version of Voronoi tessellation, made it more feasible to control morphological properties of these polyhedrons, such as cell size distribution, sphericity, and aspect ratio, by optimization of the objective function. Fundamentals of Laguerre tessellation are described as follows, and more details can be found in .n uncorrelated seeds randomly distributed within D,iX indicates seed coordinates and iW denotes the nonnegative weight coefficient at each seed point. The Laguerre element iC corresponding to each seed can be expressed as:Ld is defined as the power distance and Ed is the Euclidean distance.Given a spatial domain 4N in 3D space. A metric function is introduced by comparing the distribution X(x) of samples during the iteration with the specified probability distribution F(x) as:X(x) is a step function whose expression is:The process of adjusting seed properties to obtain the desired cell properties, herein mainly of grain size, shape, and gradations, can be transformed into an optimization problem of the objective function. The optimization variables include the seed coordinates and the square root of weight coefficient, and the number of optimization variables is Φ and a minimum absolute error of the objective function is used to terminate optimization. In this paper, the Neper program by Quey and his colleagues [Numerical integration is adopted to calculate lleagues  was adoplleagues , of whicAs the generated tessellations would occupy the complete specimen space, the mean cell size of each grading would reasonably be greater than the actual value, and the subsequent geometric smoothing and scaling adjustment are required to achieve the designed aggregate gradation of mass concrete.® . The roundness of each aggregate cell can be increased by raising the smoothness levels, or vice versa. It is worth noting that such a smoothing procedure can also remove the unnecessary parallelism between original common facets of adjacent aggregates and obtain more realistic aggregate shapes, at the cost of increasing the number of facets surrounding each cell.One can note that there are no overlaps or gaps between adjacent polyhedrons in the tessellation model, and the union of all polyhedrons occupies the whole space of a given specimen, indicating an aggregate ratio of 100%. In order to meet the requirements of 40%–70% aggregates volume content within mass concrete, and avoid the complex interparticle penetration detection process, the random convex polyhedrons generated by the Neper program in After the above geometry smoothing procedure, all the rounded aggregates are categorized into groups and numbered according to their characteristic size in an ascending order as follows:iGroup records the aggregates currently belonging to i-th group, and i,mind and i,maxd represent the minimum and maximum aggregate size of the i-th group, respectively. Take the three-graded concrete for instance, three groups are to be defined corresponding to small stones (5–20 mm), medium stones (20–40 mm), and large stones (40–80 mm). For those aggregates larger than the maximum size of all groups, they are categorized into the coarsest group. The cells after geometric smoothing that are smaller than lower bound of coarse aggregates are removed from the RAMs.Where In order to meet the size requirements, a geometric downscaling is applied with reference to the centroid of each aggregate and the dimensions of aggregates within each group can be adjusted. To fulfill the gradation or the ratio requirements of each aggregate group, a step-wise scaling procedure for the categorized aggregate groups is proposed and given below.S%, the characteristic size and volume of an aggregate after scaling are determined as follows:With a scaling factor input as Similar to the arrangement of nested sieves in order of decreasing size from top to bottom during a sieve analysis test, the proposed scaling procedure is to be conducted in a descending order, that is, from large-sized group to small-sized one. It should be noted that the aggregates belonging to the large-sized group can be downgraded into the small-sized group after a geometric reduction, hence the grouping information of all aggregates needs to be updated after a scaling treatment.i-th group, the initial scaling factor S0% is determined byi,presV is the prescribed total volume of the i-th group aggregates by the design gradation, and i,sumV is the volume value at the present stage.For the S0% given, scaling operations are looped by writing a scripting program in MATLAB with a small decrease rate, 1‰ suitable for most circumstances in practice, until the total volume of all aggregates that still belong to the i-th group is equal to i,presV A similar scaling process is followed for (i-1)-th group until all groups are scaled and the prescribed gradation is satisfied.With the initial scaling factor The generation procedures for 3D RAMs within a 450 mm-sized cubic fully graded concrete specimen is given in this section. The coarse aggregates are normally divided into four groups: 5–20 mm, 20–40 mm, 40–80 mm, and 80–150 mm. To lower the computational burden, the aggregates of the smallest-sized group, 5–20 mm, are neglected in our RAMs model, which are to be equivalently treated as the component of matrix.−6 kg/mm3 was adopted for the aggregate rock. The design volume ratio of coarse aggregates in The aggregate gradation of fully graded concrete in the testing study by Deng et al.  was chosFirstly, using the layering disposition method  providedIt can be seen that after morphological smoothing treatment, the ratio of aggregate number in each group was 1018:432:42, which was then successively adjusted by the scaling treatment of each group following a descending order of the size. After the final scaling treatment on the 20–40 mm group, the volume ratio of aggregates became 1.11:1.66:1.66, the same as that required in the testing study . Figure The above strategy simplifies the task of generating various RAM structures of concrete to a notable extent, and provides a high flexibility in the adjustment of aggregate size and gradation, as well as the volume ratio of aggregates. This section adopts the strategy for generating RAMs of typical mass concrete and presents a mesoscopic investigation into the effects of meso-structures on the overall elasticity properties. Particularly, the three-graded and fully graded concrete materials, featuring a broad range of aggregate size, were selected in this study. It is worthy to note that the present approach can be smoothly extended for the analysis of RFC, which is characterized by dense packing of extremely large aggregates over 1000 mm in size.As mentioned above, the complex shapes of aggregates would complicate the discretization process using structured or unstructured meshing techniques commonly chosen in the establishment of an FE model, and ensuring the mesh quality for the meso-structure is not an easy task. On the other hand, an automatic image-based octree meshing approach was developed by Song and his co-workers ,24, whicGenerate a stack of 2D slices from the CAD file of 3D RAM, and disintegrate the aggregate component from the matrix by the assignment of contrasting color intensities. bound to store the colors of all voxels, whose size must be integer power of 2, that is, 2n × 2n × 2n.Divide the 3D image assembled from the stack of 2D slices into cubic cells, and construct a cubic image matrix, Ibound according to the recursive algorithm by Saputra et al. [smin.Conduct the octree decomposition of Ia et al. , and theAssign material properties (aggregate or matrix) to each octree cell according to its representative color by averaging the color intensity values of internal voxels.Each octree cell is modelled as a scaled boundary polyhedral element and assembled into the system balance equation in the context of SBFEM. The nodal displacements can be solved, and in turn the stress distribution within the specimen is determined.With the CAD model of the RAMs generated, it is quite simple and direct to provide a stack of 2D slices describing the geometrical features of all coarse aggregates within a concrete sample. The residual domain, excluding the aggregates, is defined as the matrix component. It is worthy to note that when necessary, it is straightforward to incorporate the role of the interfacial transition zone by simple bolding of the boundary lines of all aggregates during the image-based generation of octree mesh. For completeness, the elementary procedures for the generation of octree polyhedral mesh and the subsequent SBFEM analysis for a cubic concrete specimen are summarized as follows, whilst more details and the theory of SBFEM can be found in Song :Generatemaxs = 8 mm and mins = 4 mm, and the maximum edge length ratio between adjacent cells was defined as not greater than 2:1, such that a balanced octree decomposition, as proposed by Saputra et al. [For all the numerical concrete samples in this study, the maximum and minimum values of octree edge length were chosen as a et al. , could bAs mentioned above, fundamental testing of concrete material demands that the specimen size is sufficiently large, as compared to the maximum aggregate size, which would bring up difficulties in the preparations of testing samples and the selections of testing machine for mass concrete with extra-large aggregates. Hence, the wet-screening method is commonly utilized in dam engineering practice by sieving out the over-sized aggregates of the fresh concrete, and the reduction of maximum aggregate size makes it feasible using small samples and the usual laboratory testing machines. Obviously, such a treatment would significantly change the size and gradation properties of aggregates in the samples as compared with the mass concrete in practice. To illustrate the effect of such a sieving procedure on the elasticity properties of concrete, three sets of UC testing specimens were generated and analyzed using the above automatic image-based approach by the SBFEM, including three-graded and fully graded dam concrete, as well as the wet-screened concrete by sieving out aggregates greater than 40 mm from the three-graded concrete batch. Five cubic specimens with random RAMs were considered in each set using the above strategy in The proportions of coarse aggregates within three-graded and fully graded numerical concrete specimens were selected according to relevant testing study ,25, in wThe elastic parameters describing the meso-level constituents within each specimen, aggregates, and matrix are given in The results in 6 mm3). A comparison study was conducted by UC modelling of all these ten specimens, and the effects of the size-reduced sampling approach in practice was investigated.For mass concrete with extremely large aggregates in practice, such as the above mentioned RFC with aggregates that may be of 1000 mm in size, the routine practice of taking specimen size two or three times greater than the maximum aggregate size poses a huge challenge to the loading capabilities of testing machines. A common compromise is to core or cut smaller samples for testing, and it is no wonder that the testing results of fundamental parameters, such as elastic modulus and uniaxial compressive strength would be influenced by such a size reduction. This section takes the above numerical sample  of fullyFrom the simulation results of the two sets of specimens, a comparison of the elastic moduli is shown in To lower the computation complexity and cost, 2D simplification is commonly adopted for meso-scale modelling of the complex behavior of concrete. It is obvious that the non-uniform spatial distribution of aggregates within actual concrete material cannot be well represented by plane modelling of typical sections. It is interesting to raise the question of whether a 2D model with coarse aggregate fulfilling the grading requirements is representative of the overall elastic behavior of concrete, or representative of the variations of aggregate distributions along slices at different portions and their effect on the evaluation of elastic properties. In this section, we generated four 450 mm-sized cubic samples of fully graded concrete to simulate the concrete batches, using a volume ratio of 52% of coarse aggregates larger than 20 mm. A thin layer of 5% of the specimen size along the outer surface of each batch was removed to minimize the boundary effects. Without loss of generality, 400 slices parallel to one surface at an interval of 1 mm were generated from the remaining cubic sample. The aggregate content of each slice was calculated and UC modelling was conducted to determine the corresponding elastic modulus. Both plane stress and plane strain analyses were adopted and the calculation results are summarized in The results in This paper presents a simple strategy for generating 3D RAMs of concrete materials, and its combination with SBFFEM and the image-based octree meshing technique allows a simple and efficient investigation into the meso-structural effects on the macro-scale mechanical behaviors of concrete. The proposed strategy firstly utilizes Laguerre tessellation for the definition of bounding boxes for the aggregates according to their size and gradation requirements, and then these tessellation cells are subjected to controlled geometric smoothing in CAD program, which can remove the unnecessary parallelism of common facets of adjacent cells and introduce a finite gap between them. The RAM generation procedure is finalized by a stepwise scaling procedure, which can efficiently tune the size and in turn the gradation of cells exactly the same as the prescribed aggregate fraction ratio. Numerical examples were given, which demonstrated the effectiveness of the proposed strategy for generating three-graded and fully graded mass concrete samples. The aggregate volume ratio can be larger than 50% and even higher. With the CAD model of the RAM generated, it is quite direct to conduct automatic image-based stress analysis using SBFEM considering the meso-level aggregate structure. Numerical studies were subsequently conducted, aiming to investigate the effects of aggregate gradation, size-reduced sampling, and 2D simplification on the evaluation of overall elasticity properties of mass concrete. The simulation results reproduced the increasing trend of the elastic modulus with the grading of maximum aggregate in literature test reports, and the simulations based on size-reduced sampling and 2D simplification can introduce relatively large scatter in results owing to the randomness of 3D aggregate distribution within a concrete specimen.The paper provides a simple and efficient strategy for the construction of random numerical samples of concrete, considering high aggregate content and broad range of gradation. With additional work of compaction simulation, it can be extended for the simulation of dense packing of extremely large aggregates in RFC, of which the size may be greater than 1000 mm and the aggregate volume ratio can be as large as 60%. Moreover, the automatic image-based SBFEM modelling is proven to be appropriate for meso-level simulation of the elasticity properties of mass concrete, and can be extended for nonlinear damage and failure analysis under this framework.


Concrete barely possesses tensile strength, and it is susceptible to cracking, which leads to a reduction of its service life. Consequently, it is significant to find a complementary material that helps alleviate these drawbacks. The aim of this research was to determine analytically and experimentally the effect of the addition of the steel fibers on the performance of the post-cracking stage on fiber-reinforced concrete, by studying four notch-to-depth ratios of 0, 0.08, 0.16, and 0.33. This was evaluated through 72 bending tests, using plain concrete (control) and fiber-reinforced concrete with volume fibers of 0.25% and 0.50%. Results showed that the specimens with a notch-to-depth ratio up to 0.33 are capable of attaining a hardening behavior. The study concludes that the increase in the dosage leads to an improvement in the residual performance, even though an increase in the notch-to-depth ratio has also occurred. Concrete is the most used construction material around the world due to its versatility, formability, and the widespread availability of its ingredients ,2,3. HowConsequently, material science and concrete technology have strived to find a complementary material that allows for alleviating these drawbacks and, once the first-crack load is attained, to provide the ability to prevent complete fracture. In other words, they are striving to find a material that favors the improvement on strain capacity and energy absorption.One of these materials is steel fibers. Several studies with steel fibers have proven a minimum improvement in the concrete compressive strength ,11,12,13Steel fibers are suitable for reinforcing concrete since they provide an energy dissipation mechanism and control the crack propagation in residual stages more efficiently than plain concrete. This latter is one of the main advantages in the use of this reinforcing material. However, other characteristics can be affected due to the effective concrete area, for instance, the residual performance of the fibers, which is one of the primary factors in the transmissibility of stresses through the bond developed between the fiber and the cementitious matrix .The study of the residual performance is possible through notched specimens with controlled-induced crack, from where representative data are obtained, and for which the dispersion coefficients are less than those for un-notched specimens ,23.Several advantages have been reported on the use of steel-fiber-reinforced concrete (SFRC), for instance, the reduction in terms of costs and an improvement in the quality of structures, mainly in crack control and under cyclic loading . Some auRegarding bending tests for characterizing fiber-reinforced concrete, it has been found that the failure mode of the specimen is influenced by its span-to-depth ratio; this means that its failure mode is governed by shear or bending .For the residual strength assessment, international codes and standards have proposed three-point bending tests on beam specimens notched at midspan, to control the crack development, by establishing a notch-to-depth ratio  of 0.16 .3 (0.25%) (Series 1) and another of 40 kg/m3 (0.50%) (Series 2), in specimens subjected to flexural tension  on prismatic specimens of 150 mm × 150 mm × 600 mm. The crack initiation was controlled by inducing a notch in the specimens [In this study, the effect of the notch-to-depth ratio in the fiber-reinforced concrete was studied, by utilizing two dosages of steel fibers added, one of 20 kg/mpecimens ,18,29 ofThe novelty of the present research lies in the determination of the residual performance of the fiber-reinforced concrete for notch-to-depth ratios (a/d) lower than 0.16; this differs from other studies found in the literature where larger notch-to-depth ratios are considered. The consideration of lower ratios allows for the study of the post-cracking behavior and the fracture energy in a fracture process zone larger than that of the standards.To understand the influence of the (a/d) ratio, it is necessary to mention some of the concepts of nonlinear fracture mechanics in concrete. In general terms, fracture mechanics is defined as a failure theory of great utility, since it utilizes energetic criteria that, together with strength criteria, account for the propagation of the crack through the structures . HoweverThe nonlinear fracture mechanics is one of the theories that better describes the concrete behavior; nonlinear fracture mechanics is the study of the cracking of solid bodies that show a nonlinear constitutive response in nature, contrary to LEFM, where geometrical and material linearity is considered .The construction materials can show several behaviors depending on the applied loads; some materials can manifest barely or null deformation capacity, so they are considered to be fragile materials. On the contrary, there exist materials considered to be ductile. Concrete is a special case, as its behavior cannot be characterized completely as fragile, since it is more likely described as a quasi-fragile material. Concrete shows a gradual decay of the tensile stress (strain softening) with or without an improvement on the tensile strength, after the development of the first crack (strain hardening); hence, in general terms, the failure can be presented without yielding ,33.Another important aspect to consider is the capacity of energy absorption, which is obtained as the area under the curves of load-displacement or load-crack opening. The consideration of energy absorption is necessary, especially under dynamic loading, since it determines the ductility of the structure. In fragile materials, the elastic energies are dissipated as superficial energy, without an FPZ. Meanwhile, in ductile materials, the FPZ is a plastic zone that can dissipate a large amount of energy, larger than the superficial energy; for quasi-fragile materials, such as concrete, the FPZ is usually larger than the zone for ductile or fragile materials ,35 and dThe addition of steel fibers to the concrete, in a determined fraction of volume, improves ductility and increases the initial width of the FPZ, which gives, as a result, the enlargement of the zone due to the extraction of the fibers . The steIn the same manner, the use of steel fibers largely increases the energy absorption and ductility . It is iIn comparison with plain concrete, this material shows an extended softening branch that is characterized by a significant tensile residual strength and higher fracture energy , with thFor this research, 4 plain concrete mixtures were produced, one for each notch-to-depth ratio used. Furthermore, 8 mixtures of fiber-reinforced concrete (4 for each fiber percentage used) were also produced. The mixtures were produced by using cement OPC 40, which satisfies NMX-C-414-ONNCCE-2014 ; standarf) of 50 mm, a diameter (df) of 1 mm, an aspect ratio (lf/df) of 50, and a tensile strength of 1130 MPa; the fiber volumes were 0.25% and 0.50%, which are the volumes recommended to obtain a residual strength according to the standard EN 14845-1, 2007 [3 and a water/cement ratio of 0.55. The composition for each mixture is shown in The steel fibers utilized were “hooked-end” fibers, with a length . The first mixture was required as the reference series . From this mixture, 6 beams of 150 mm × 150 mm × 600 mm for each depth were fabricated; thus, a total of 24 specimens were constructed.In the second and third mixes, volumes of fibers (Vf) of 0.25% and 0.50% were used, respectively. Consequently, a total of 24 specimens were also constructed for each mix and for each notch depth. Hence, a total of 48 prismatic specimens were constructed with fibers, with the dimensions and notch depths aforementioned.The effect of the addition of steel fibers on the post-cracking performance was evaluated in the 72 specimens (plain concrete and fiber-reinforced concrete), through a flexural tension test of the notched beams with notch depths previously described, and with their corresponding notch-to-depth ratio (a/d) of 0, 0.08, 0.16, and 0.33, respectively. It is worth mentioning that the notching procedure was carried out according to the standard EN 14651-2005, where the common notch depth is 25 mm [In this experimental program, the crack-mouth-opening displacement method (CMOD) was used, plotted against the applied load. The measurements of the openings were conducted by employing clip-on-type extensometers, with a gauge length of 20 mm and a stroke of +12 mm/–2 mm see a, with tL = stress at the proportional limit (N/mm2), FL = load at the proportional limit (N), L = span of the specimen (mm), b = width of the specimen (mm), and hsp = distance between the top face of the specimen and the tip of the induced crack (mm).Experimental results were obtained from the load at the proportional limit or from the load for the first crack (FL), which is the larger value of load recorded up to a CMOD of 0.05 mm ,36. The Rj), at each specific value of the measured CMOD, and computed by Equation (2) [R,j = normal residual stress at the point j (N/mm2), fRk,j = characteristic residual stress at point j (N/mm2), and FR = load for a given crack-mouth opening displacement measured.The contribution of the steel fibers is of major importance at the residual stage. This contribution, at the post-cracking stage, is obtained through the normal stresses (ftion (2) .(2)fR,jR,1, fR,2, fR,3, and fR,4, are the normal residual stresses (N/mm2) for a 0.5, 1.5, 2.5, and 3.5 mm crack-mouth opening, respectively. In a similar manner, the values of fRk,1, fRk,2, fRk,3, and fRk,4 are the characteristic residual stresses (N/mm2) for a 0.5, 1.5, 2.5, and 3.5 mm crack-mouth opening, respectively.The values of fxN = statistical factor when the coefficient of variation of the population set is known, and kxn = statistical factor when the coefficient of variation of the population set is unknown.In this research, the assessment of the characteristic residual strength was obtained according to the procedure established in CEB-FIP model code 2010 , throughTwo models were utilized in the estimation of the fracture energy in the fiber-reinforced concrete specimens, where their respective parameters and principles differ from each other. For model 1, proposed by Barros et al. , shown if = area under the curve load vs crack opening (or displacement), work due to fracture (N-m) or (J); m = mass of the specimen (kg); 2); 2); L = length of the specimen (mm); and r = distance between the top face of the specimen and the crack tip (m).Model 2, proposed by Kazemi et al. , is presIn the same manner, in The required stresses for the first crack to appear, for each studied series, are presented in The least dispersion in the results was obtained for Series 1 and 2, with a ratio of (a/d) = 0.16, which is the notch-to-depth ratio suggested by the standard EN 14651, 2005 [In the figures corresponding to Series 2, a larger performance in the post-cracking stage is shown, in comparison with those corresponding to Series 1, reaching a residual load equal to, or even larger than, the average load obtained in the first crack-occurrence stage. This results in a hardening behavior due to the larger amount of fibers used , which improves the performance in the post-cracking stage by increasing the capacity to transfer stresses through the cracked faces.By analyzing the results of both series of the obtained curves, in R,j) are shown, for both studied series. Based on the (a/d) ratios utilized, it can be noticed that, from In In the same manner, the maximum performance was reached with the increment on the dosage of fibers for the ratios between 0.08 and 0.16, while for 0.33, its residual performance decreased.Rk,j) for each (a/d) ratio, for both Series 1 and 2, are shown, along with their corresponding average stress curves for each case. It can be noticed that the characteristic residual stresses are significantly sensitive to the variation of the normal residual stresses; this can be verified in In For Series 1, by increasing the ratio (a/d), a more suitable performance in the post-cracking stage was attained, this suggests that there is an adequate transmission of stresses on the faces of the specimens. On the other hand, in However, the behavior with respect to the ratios (a/d) of Series 2 is not consistent with what was obtained in Series 1, since the ratio (a/d) = 0.08 provides the best residual performance of all the ratios utilized see b. This cx, and, thus, the variation in the results will be lower.The assessment of the characteristic residual stress close to the average normal stress, obtained from experiments, requires a lower dispersion between tests. This can be attained from the increment on the number of specimens. This will result in a decrease in the statistical uncertainty factor, kxn = 2.33 and 2.18 were used, for the series where five and six specimens were tested, respectively. The (a/d) ratios that do not show any classification are those that did not attain the minimum characteristic residual strength of 1 N/mm2. In The results obtained for the ratios (a/d) = 0 and 0.08 of Series 1 are low and insufficient in order to be able to reach the minimum characteristic residual stress and to be able to classify their residual behavior. On the contrary, the ratios (a/d) = 0.16 and 0.33 showed perfectly plastic behavior and a hardening behavior, in their respective residual response.The results for Series 2, as shown in 3), the fracture energy increases even if the (a/d) ratio increases. This behavior implies that, due to the presence of a larger amount of fibers, the cracking strength increases by means of a bridging mechanism of the stresses throughout the cracked faces, which can generate a multiple cracking condition in the matrix.The increase in the (a/d) ratio for Series 1 has the adverse effect in the fracture energy, as computed with the fracture work model 1, as it is observed in In this manner, the fracture energy obtained in the post-cracking stage will be influenced by the amount of fibers located in the cement matrix and by the capacity of these to transfer stresses in the residual stage, since the extraction process of the steel fibers generates a higher consumption of energy due to the straightening of their hooked end. These will also have the same capacity of diminishing the stress concentration in the upper end of the crack, by limiting the crack propagation and also by limiting the cracking occurrence ,55,56,57As formerly mentioned, the fracture energy indicates an important contribution of the fibers in the residual stage, given that the average increment of the fracture energy for Series 2 was of more than 100%, for the ratios (a/d) = 0.08 and 0.33, with respect to those results obtained for Series 1. For the ratio (a/d) = 0, the difference is only 57%; this indicates that the notch absence in the specimen does not allow for the efficient behavior of the fibers, thus limiting the increase of energy fracture.In the graphs, the necessity of zero energy is considered to fracture beams whose notch goes from top to bottom; this means that the curve is assumed to start at the origin. Furthermore, the slope of the curve represents the energy consumed for the crack to grow a unit in depth. By dividing this slope by the width of the beam (b), it gives as result the fracture energy required.It is worth mentioning that the results of the fracture energy obtained through this model are relatively close to those obtained by the first model. Such values are represented in 2 will also increase. This may lead to a problem to obtain representative results for the behavior of the post-cracking stage of the composite material.It can be observed that the second model has a direct relation with the variation of the results, since, by obtaining high variation coefficients, the factor RThe results presented in this article are limited to fiber-reinforced concrete with steel fibers, and with the percentage of volume fibers already described. Therefore, experiments with other types of fibers  and with different characteristics are desirable to extend the range of opportunities in the improvement of the post-cracking response of the concrete.The load at the proportional limit is not affected by the addition of steel fibers, given that the obtained values of load were closed in each of the studied series. This indicates that, for this stage, the performance of the material depends mostly on the cement matrix and the remaining concrete area.The load and the stresses at the proportional limit exhibited an inversely proportional behavior to the notch-to-depth ratio, where an increase in such ratio will result in the concrete to be prone to failure.The increase in the dosage of fibers leads to an improvement in the normal and characteristic residual stresses.For Series 2, the increase of the notch-to-depth ratio (a/d) enhances the normal and characteristic residual performance. For the ratio (a/d) = 0, the increment in the normal residual stress was 61%, while for the ratios (a/d) = 0.08, 0.16, and 0.33, the increment was 157%, 129%, and 86%, respectively. The ratio (a/d) = 0.08 provides the best characteristic residual performance of all the ratios considered.For Series 1, the notch depth of 25 mm, equivalent to a ratio (a/d) of 0.16, was the only ratio that met the minimum residual stress requirements established in international standards.3) and for low (a/d) ratios , it was not possible to reach the minimum classification of residual strength, due to the fact that the specimens were unable to reach the minimum values.For a low amount of fibers  = 0.33, which implies that the performance in the post-cracking stage does not depend on the concrete, but rather on the capacity of the fibers to transfer stresses through the cracked faces of the specimen and also on the amount of fibers located in an analyzed section.3. This implies that the steel fibers contribute to improving the residual performance of the composite material.The fracture energy increased in about 97% (model 1) and in about 35% (model 2), by increasing the volume of fibers from 20 to 40 kg/mFor Series 2, the fracture energy increased even if the (a/d) ratio also increased. The presence of a larger amount of fibers allows the cracking strength to be incremented and to generate a multiple cracking condition in the matrix.The mathematical models used showed similar results, particularly for high contents of steel fibers in the concrete.The results obtained in this research will offer an experimental frame of reference for different ratios (a/d), with respect to the one recommended in the standard, which can facilitate having a criterion of analysis with respect to the residual stresses determined from laboratory tests.Based on the results, the following conclusions can be drawn:


Trichophyton mentagrophytes, a zoophilic species, is one of the most frequently isolated dermatophytes in many parts of the world. This study investigated the efficacy of a sophorolipid (SL-YS3) produced by Rhodotorula babjevae YS3 against dermatophytosis caused by T. mentagrophytes. SL-YS3 was characterized by gas chromatography–mass spectrometry (GC–MS) and ultra-performance liquid chromatography, coupled with electrospray mass spectrometry (UPLC-ESI-MS). SL-YS3 comprised of six different fatty acids as the hydrophobic components of constituent congeners and sophorose as the hydrophilic component. Inhibitory effects of purified SL-YS3 against hyphal growth was found to be 85% at a 2 mg ml–1 concentration, and MIC was 1 mg ml–1. Microscopic examination with scanning electron microscopy (SEM), atomic force microscopy, and confocal laser scanning microscopy (CLSM) revealed that SL-YS3 exerts its effect by disrupting cell membrane integrity causing cell death. SL-YS3 was also effective in reducing the biofilms formed by T. mentagrophytes, which was observed spectrophotometrically with crystal-violet staining and further validated with SEM and CLSM studies of treated biofilms. In vivo studies in a mouse model of cutaneous dermatophytosis involving macroscopic observations, percent culture recovery from skin samples, and histopathological studies showed that SL-YS3 could effectively cure the infected mice after 21 days of topical treatment. Terbinafine (TRB) was used as a standard drug in the experiments. We demonstrate, for the first time, the antidermatophytic activity of a sophorolipid biosurfactant. The findings are suggestive that SL-YS3 can be formulated as a novel antifungal compound to treat cutaneous mycoses caused by T. mentagrophytes. Trichophyton mentagrophytes is a zoonotic dermatophyte found in a variety of environments and is especially dominant in clinical cases of tinea pedis and onychomycosis, with a prevalence of 1.5–55.4%  in the SQLE gene resulting in high MICs. Similarly, T. interdigitale and T. rubrum isolates. In addition, T. rubrum was linked to the salA gene, encoding salicylate 1-monooxygenase, where increased expression of salA resulted in resistance. Thus, there is a need for the search of new sources of anti-dermatophytic compounds, which are efficacious, cheaper, and safer/non-toxic alternatives to their synthetic counterparts. Despite the high incidence of dermatophyte infections, few investigations have been performed to better understand these infections, and most of the studies were based on in vitro experiments  was obtained from the National Culture Collection of Pathogenic Fungi (NCCPF), India. The fungus was cultured in Sabouraud Dextrose broth/agar , and the microconidial suspension was stored at −80°C in 30% glycerol (v/v) for maintenance. The working cultures were maintained in SDB/SDA plates at 4°C and subcultured every 2 weeks.In the present study, the yeast strain er study . The strRhodotorula babjevae YS3 was cultured in Bushnell-Haas medium  at 19 ± 2°C with 10% (w/v) glucose as the carbon source for the production of SL (SL-YS3) used in this study . Helium was used as the carrier gas at a flow rate of 1 ml min–1. MS scan was performed in the range of m/z 10-350 and analyzed with GC–MS solution software (version 4). Compounds were identified using the NIST 11 library database.is study . A detaiis study . Additiois study . Briefly18 selectivity column  using a gradient of water + 0.1% formic acid (solvent A) and acetonitrile (solvent B) as the mobile phase at 40°C at a flow rate of 0.2 ml min–1. MS scans were performed in the scan range of m/z 66–1000 and analyzed using Thermo Scientific Xcalibur software (version 2.3.0.1765). During both GC–MS and UPLC-ESI-MS analyses, a commercially available SL, 1,4′′-sophorolactone 6′,6′′-diacetate  was used as the reference standard.The monosaccharide composition of the sugar moiety in SL-YS3 was analyzed by UPLC-ESI-MS  . SL-YS3 T. mentagrophytes against SL-YS3 was tested using the broth microdilution method as per the Clinical and Laboratory Standards Institute (CLSI) M38-A2 reference for filamentous fungi (4 to 5 × 104 CFU ml–1). SL-S and terbinafine  were used as reference standards. Double strength stock solutions of the test compounds were prepared, and serial twofold dilutions were made in RPMI-1640 medium buffered to pH 7.0 with 0.165M 3–(N–morpholino) propanesulfonic acid (MOPS) buffer . A 100 μl of the prepared conidial suspension was added to each well of 96-well microtiter plates containing 100 μl of test compounds. Growth and sterility controls were also included, and the plates were incubated at 25°C for 96 h. The absorbance of each well was measured at 600 nm using a Multimode Reader (Thermo Scientific). The concentration showing no growth was considered as the minimum inhibitory concentration (MIC).Antifungal susceptibility of us fungi . The conT. mentagrophytes in SDA was transferred to fresh plates containing SL-YS3 (0.5 to 4 mg ml–1) and incubated at 25°C  and TRB  for 7 days at 25°C, and then subjected to microscopic observations. Mycelia without any treatment served as a negative control.Mycelia of For scanning electron microscopy (SEM), mycelia were excised and fixed in 2.5% glutaraldehyde (v/v) in phosphate-buffered saline (PBS) overnight at 4°C. After washing the samples thoroughly in PBS to remove the excess fixative, they were dehydrated in graded ethanol series (30–100% v/v) for 15 min each. The samples were then mounted on stubs over carbon tape and sputter-coated with gold-palladium and observed in a field-emission scanning electron microscope  .Mycelial topography was studied in semi-contact mode using an atomic force microscope , equipped with a silicon cantilever (spring constant 0.03 N/m) and a scanner (100 μm × 100 μm × 5 μm scan range). Images were analyzed with Nova software version 1.1.0.1780.–1 propidium iodide  for 15 min, washed in PBS, and visualized in a confocal microscope  , the treated mycelia were excised and then stained with 0.1 mg mlGermany) .Trichophyton mentagrophytes biofilms were formed in 96-well flat-bottom microtiter plates, according to 6 CFU ml–1) was prepared in RPMI-1640 medium and inoculated in appropriate wells except in those for sterility control. After incubation at 28°C for 96 h, the medium was decanted, and the wells washed thoroughly with sterile PBS to remove any remaining planktonic cells. Serially double diluted solutions of SL-YS3 and TRB (standard drug) prepared in RPMI-1640 were added to the biofilms and incubated for 24 h. Control wells contained only 100 μl RPMI-1640 without SL-YS3 or TRB. After incubation, the wells were washed with PBS, and 100 μl of 0.5% crystal violet (CV) solution was added at room temperature to stain the biofilms for 15 min. The wells were washed with sterile water to remove excess stain, and biofilms were decolorized by adding 100 μl of 95% ethanol solution in each well. The ethanol solution was gently homogenized with a pipette until the rest of the CV was completely dissolved (∼1 min). Finally, the solution from each well was transferred to a new 96-well plate and then read in a Multimode Reader at 570 nm. The antibiofilm activity was calculated as:T. mentagrophytes, as described previously  glutaraldehyde in PBS  at 4°C. After the PBS washes, to remove any traces of fixative, the samples were dehydrated in an increasing gradient of ethanol (30–100% v/v), mounted on stubs over carbon tapes, metalized with gold-palladium, and observed in a field emission scanning microscope .–1 propidium iodide , for 15 min and viewed in a confocal microscope .For CLSM, fungal biofilms post-treatment were gently washed with PBS, stained with 100 μg mlad libitum.Equal numbers of male and female Swiss albino mice  were randomly selected and maintained in the animal house facility at IASST under a controlled temperature (22 ± 2°C), with relative humidity (60–70%), and a 12:12 h light–dark cycle. The experimental protocol was approved (IASST/IAEC/2016-17/05) by the Institutional Animal Ethics Committee (IAEC) of IASST, Guwahati before starting the experiments and was performed using the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India, on animal experimentation. Mice were individually housed in polypropylene cages and fed a rodent pellet diet  and water –1) and xylazine (10 mg kg–1)  TRB; Group IV – topical treatment with 1 mg ml–1 (w/v) SL-YS3. After 72 h (after the establishment of infection), treatment was initiated once a day and was continued for 21 days.For induction of trichophytosis, animals were anesthetized with a cocktail of ketamine (80 mg kgmg kg–1) . Dorsal Skin scrapings were collected from each infected site at intervals of 7 days, cultured in SDA plates, and incubated at 25°C for 7 to 8 days. The skin scrapings that yielded fungal growth were considered to be culture positive, and animals with one or more culture-positive skin scraping was considered to be fungus positive .Culture recovery was calculated as:For evaluation of the histopathological changes, the animals were euthanized by cervical dislocation at the end of the 21-day treatment period, and skin biopsy samples were fixed in neutral buffered formalin. Paraffin-embedded tissues were sectioned (4–5 mm) and stained with hematoxylin and eosin (H&E) and Periodic acid-Schiff (PAS) stain for light microscopy. The pathological changes were examined under a light microscope .in vitro antifungal effect of SL-YS3 and culture recovery (%) from the treatment groups of the in vivo experiments were statistically validated using a one-way analysis of variance (ANOVA) followed by the pairwise least significant difference (LSD). Differences were considered significant at P < 0.05. Statistical analyses were performed using SPSS Statistics v22.0 .All the experiments were conducted with at least three replicates and repeated once. The results are expressed as mean ± SD. The + of glucose. The base peak at m/z 149 could be attributed to the fragment ion generated after the loss of a hydroxymethyl group (M-CH2OH), while the small peak at m/z 365 represented the monosodiated glucose dimer adduct after the loss of a water molecule [2M + Na-H2O]+.The UPLC chromatograms of the sugar component (hydrophilic moiety) of both the samples yielded a single peak having the same retention time . Subsequ–1, respectively (–1). At the highest concentration of SL-YS3 tested, i.e., 4 mg ml–1, complete inhibition of mycelial growth was recorded.Broth microdilution assay was performed for the initial characterization of the antifungal activity. SL-YS3, SL-S, and TRB exhibited complete inhibition of spore germination (MIC) at concentrations 1.0, 1.0, and 0.031 mg mlectively . Figure T. mentagrophytes post-treatment with SL-YS3 and TRB. Severely damaged mycelia with irregular morphology, flattened hyphae along with ruptured surfaces were observed. On the other hand, untreated control hyphae had a uniform cell morphology and no visible damage.Atomic force microscopy was carried out to understand the topographical alterations of the fungal cells being treated with SL-YS3. Confocal laser scanning microscopy was performed to determine the cell membrane damage as measured by the uptake of PI. In the control samples, no fluorescence could be detected. However, after exposure to SL-YS3 and TRB, increased fluorescence was observed, indicating the uptake of PI due to loss of cell membrane integrity .F4,40 = 184.621, P < 0.000). Although the biofilms were not completely eradicated at 2 × MICs of SL-YS3 (2 mg ml–1) similar to TRB (0.062 mg ml–1) and SL-S (2 mg ml–1), a significant reduction  was noted.The effect of a range of concentrations of SL-YS3 on the disruption of biofilms formed on polystyrene microtiter plates was evaluated with CV staining . SL-S anT. mentagrophytes biofilms and distinct structural differences on exposure to SL-YS3 and TRB in comparison to the untreated control. SEM micrographs  with comparable efficiencies . The complete eradication of the fungus was evident from the absence of culture-positive skin tissue collected at the end of the 21-day treatment period  and hydrophilic moiety (sugar component), respectively. In GC–MS analyses of the FAMEs, it was revealed that SL-YS3 comprised of congeners containing six different fatty acids. The most abundant fatty acids in SL-YS3 were palmitic acid (C16:0), stearic acid (C18:0), and lauric acid (C11:0). The stearic acid side chain was also observed in the SL-S analyzed under the same set of conditions and exhibited a similar retention time and mass spectrum. The UPLC-ESI-MS analyses of the sugar components of both the samples confirmed glucose as the sole monomer present, and no other sugars were detected, confirming SL-YS3 to be an SL type of biosurfactant. The mass spectral analyses substantiated the same upon comparison with previous literature  as reportedly, the presence of acetyl groups enhances the biological activity of SLs , and increased fluorescence in the treated samples (CLSM). Compounds that can remove the ECM may be an effective strategy to disperse biofilms, disaggregate the cells, and disrupt the pathogenic environment. This might be due to the surfactant action of SL-YS3. Recalcitrance and resistance have been frequently observed in dermatophytic infections, especially onychomycosis frequently caused by T. mentagrophytes that have been related to the formation of biofilms  of IASST, Guwahati.SS and SD designed the entire study. SS carried out all the experiments and wrote the manuscript. SB helped with the experiments, analyzed the statistical data, and contributed to the manuscript writing. RK helped in the design and execution of the animal model. AB provided valuable input during the experimental design. All the authors have reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


While several studies have assessed knowledge, attitudes and behaviours of the public, physicians and medical students in a number of EU/EEA countries with respect to antibiotic use and antibiotic resistance, there is a paucity of literature for other healthcare workers. This survey aimed to fill this gap.A 43-item online questionnaire was developed, validated and pilot-tested through a modified Delphi consensus process involving 87 Project Advisory Group (PAG) members, including national representatives and members of European health professional groups. The survey was distributed by the PAG and via social media to healthcare workers in 30 EU/EEA countries.Respondents  from 30 EU/EEA countries participated. Knowledge of antibiotics and antibiotic use was higher (97%) than knowledge of development and spread of antibiotic resistance (75%). Sixty percent of respondents stated they had received information on avoiding unnecessary prescribing, administering or dispensing of antibiotics. Among respondents who prescribed, administered or dispensed antibiotics, 55% had provided advice on prudent antibiotic use or management of infections to patients, but only 17% had given resources (leaflets or pamphlets). For community and hospital prescribers, fear of patient deterioration or complications was the most frequent reason (43%) for prescribing antibiotics that were considered unnecessary. Community prescribers were almost twice as likely as hospital prescribers to prescribe antibiotics due to time constraints or to maintain patient relationships.It is important to move from raising awareness about prudent antibiotic use and antibiotic resistance among healthcare workers to designing antimicrobial stewardship interventions aimed at changing relevant behaviours. In Europe, 33,000 people die from infections with antibiotic-resistant bacteria each year. An estimated EUR 1.5 billion is spent annually on healthcare costs and loss of productivity due to antibiotic resistance . The cauWhile previous multi-country and European Union (EU)-wide studies have focused on the public‘s -7, physiThis is a baseline study, which aims to: (i) assess the knowledge, attitudes and behaviours of healthcare workers in 30 EU/EEA (European Union/European Economic Area) countries with respect to antibiotics, antibiotic use and antibiotic resistance; (ii) provide a baseline dataset for designing and evaluating future policy, communication and educational interventions; and (iii) support the evaluation of awareness raising campaigns including EAAD.In October 2018, ECDC National Focal Points for Antimicrobial Resistance and National Focal Points for Communication from all EU countries, two EEA countries (Iceland and Norway), and selected European health professional organisations or groups, were invited to designate representatives to participate in the present study as members of a Project Advisory Group (PAG). The PAG comprised 87 individuals representing the EU/EEA countries and European professional organisations listed at the end of the article.Many models of behaviour change have been used to understand and predict health behaviour. Examples include the theory of reasoned action, the health belief model, social learning theory, and the trans-theoretical stage model ,16. We sA 43-item web-based questionnaire was developed following a systematic review of the literature and a two-round Delphi consensus process with members of the PAG. The questionnaire was pilot tested in the participating EU/EEA countries, and validated after translation into the 24 EU official languages, Icelandic and Norwegian. Further questionnaire details are available in Supplement 1 EU EEA antibiotic survey questionnaire with answer options, and Supplement 2 mapping of survey questions to COM-B.The questionnaire included multiple choice questions, statements testing knowledge using a true or false answer, and statements assessing attitudes and behaviours by seeking agreement using a 5-point Likert scale – strongly agree, agree, neither agree nor disagree, disagree, strongly disagree. In addition, there was an option of ‘I do not understand the question/not applicable’. Further details are available in Supplement 2 Mapping of survey questions to COM-B. The questionnaire consisted of ten sections .BoxDemographic questions: for each respondent to fill in at the beginning of the survey.Capability: perceived and actual knowledge on human, environmental and animal health factors.Opportunity: level of access to guidelines for managing infections, access to materials to give advice on prudent use of antibiotics and AMR, and questions determining how often they gave out resources and advice.Motivation: level of agreement/disagreement with personal role in helping to control antibiotic resistance, and the connection between their prescribing OR dispensing OR administering of antibiotics and emergence and spread of antibiotic resistant bacteria.One health: level of agreement or disagreement with statements on whether specific environmental and animal health factors contribute to antibiotic resistance.Hand hygiene: self-assessment on being able to list the WHO’s five moments of hand hygiene and whether they needed to perform hand hygiene (as often as recommended) if wearing gloves.Information available on antibiotic use and antibiotic resistance or managing infections: recollection of receiving information about avoiding unnecessary prescribing OR administering OR dispensing of antibiotics, and source(s) of information that had most influenced their views on antibiotic use and resistance.Campaign  and training: level of awareness of EAAD and World Antibiotic Awareness Week (WAAW), and the perceived effectiveness of these campaigns in raising awareness about prudent use of antibiotics and antibiotic resistance within the respondent’s country.Future contact: to determine whether respondents wanted the project team to contact them about their survey responses or other relevant AMR activities, and a question on how they found out about the survey.Question for prescribers: level of confidence in making antibiotic prescribing decisions, access to antibiotic guidelines and confidence in the antibiotic guidelines available to them, individual role in controlling antibiotic resistance, and how often they prescribed antibiotics in the previous week when they would have preferred not to.AMR: antimicrobial resistance; EAAD: European Antibiotic Awareness Day; EU/EEA: European Union/European Economic Area; WHO: World Health Organization.A quota sampling approach was used to determine the minimum survey sample size required with the aim of generating a representative sample from different healthcare worker groups in the participating countries. The overall sample size for the study and the sample size per country was determined by calculating 0.2% of all practicing physicians, dentists, pharmacists and 0.1% of all nursing professionals, registered in healthcare personnel statistics for each country individually, and combined from the EU/EEA . The proThe validated online questionnaire was distributed by the PAG members to healthcare workers in their country, and promoted via social media using #ECDCAntibioticSurvey. Participation was voluntary, and the online questionnaire was open for responses over a 6-week period between 28 January and 4 March 2019. All healthcare workers in each of the 30 participating EU/EEA countries were eligible to complete the online questionnaire.Data were collected anonymously. All data were held securely in Public Health England’s internal networks and in line with the General Data Protection Regulation 2016/679.Descriptive statistics for frequency distribution and percentages were used to analyse the respondents’ knowledge and understanding. Comparisons were made using the chi-squared test, and associations were assessed using the odds ratio. A five-point Likert scale was used for several questions and the ‘agreed’ and ‘strongly agreed’ responses were merged and reported as ‘agreed’.Data were analysed using MS Excel (2010) and STATA statistical software release 15 ). Level of significance was set to p < 0.05.All respondents participated strictly in their professional capacity, and were provided with informed consent prior to participation, according to the Declaration of Helsinki.Further details of the cross-sectional survey using the CHERRIES checklist for web-based studies is available in Supplement 3 Cherries checklist for ECDC antibiotic survey .The estimated required quota sample size was 11,931 respondents. In total, 18,365 healthcare workers from the 30 EU/EEA countries responded to the survey, thus exceeding the required quota. Overall demographic data including respondents’ age, gender, years in current profession and professional setting they work more than 50% of the time for the 30 EU/EEA countries are presented in Twenty-four of 30 countries achieved or exceeded the required quota sample size of respondents, two countries achieved more than 70% of their quota sample size of respondents (77% and 84%), while four countries achieved less than 60% of the required quota sample size. The number of responses from medical doctors , dentists and pharmacists substantially exceeded the required quota sample size for these professions (Supplement 4 Table 1). The number of responses from the nursing profession was only slightly higher  than the required quota sample size , and only 55% of the required quota sample size for other healthcare workers was achieved.Eighty-two percent of respondents  stated that their role involved interacting with patients or members of the public in one or more of the following capacities: diagnosing, prescribing, clinical checking of prescriptions, dispensing, administrating, or providing advice on antibiotics . Only 28Ninety-six percent of respondents agreed with the statement ‘I know what antibiotic resistance is’, and 80% agreed with the statement, ‘I have sufficient knowledge about how to use antibiotics appropriately for my current practice’. Responses varied by healthcare worker group (range 44 – 87%)  and by cFour of the seven knowledge test statements ‘Antibiotics are effective against viruses’, ‘Antibiotics are effective against cold and flu’, ‘Taking antibiotics has associated side effects or risks such as diarrhoea, colitis, allergies’ and ‘Unnecessary use of antibiotics makes them become ineffective’ were correctly answered by more than 90% of respondents. Two statements ‘Healthy people can carry antibiotic resistant bacteria’ and ‘Antibiotic resistant bacteria can spread from person to person’ were answered correctly by more than 80% of respondents. The statement ‘Every person treated with antibiotics is at an increased risk of antibiotic resistant infection’ was correctly assessed as true by only 75% of respondents, the lowest proportion of the seven questions .There was a variation in the percentage of respondents answering all seven key knowledge questions correctly across the thirty EU/EEA countries (40%–73%) and professions (29%–58%) (Supplement 4 Tables 3 and 4). No country had 100% of respondents who achieved seven of seven correct answers in the knowledge score, however, most countries (21/30) had more than 50% of respondents answer all the key knowledge questions correctly. Overall, 58% of respondents answered all seven questions correctly, with an average score of 6.35/7 across the 30 EU/EAA countries. Substantial variation was noted between countries (Supplement 4 Table 3). Medical doctors answered all the questions correctly more often than any other respondent category (68%) , followed by pharmacists (59%) (Supplement 4 Table 4).Findings from the knowledge test questions on environmental and animal health factors and on hand hygiene are provided in Supplement 4 Figures 1 and 2. Only 27%  of respondents correctly identified that it is illegal in the EU to use antibiotics to stimulate growth of farm animals; 44%  were unsure and 29%  believed it was legal practice. Just over half of respondents 56%  stated that they could list the World Health Organization‘s five moments for hand hygiene .Seventy-five percent  of respondents with direct patient or public involvement agreed that they had easy access to guidelines on managing infections, 68%  agreed they had easy access to materials for advising patients about prudent antibiotic use and antibiotic resistance, and 72%  agreed that they had good opportunities to provide advice about antibiotic use. Substantial variation was noted by country, profession and setting .Ninety-two percent of respondents  with direct patient contact agreed that they knew there was a connection between their prescribing/dispensing/administering of antibiotics and the emergence and spread of antibiotic-resistant bacteria, but only 63%  agreed that they have a key role in helping control antibiotic resistance. The proportion of respondents who agreed that they have a key role in helping control antibiotic resistance was higher for those who worked in community settings (65%) than those in hospitals (56%) and other settings (55%).Sixty-five percent  of all respondents had either prescribed, administered or dispensed antibiotics at least once in the week prior to completing the survey. Of these respondents, 17%  had given resources (e.g. leaflets or pamphlets), and 55%  had provided advice on prudent antibiotic use or management of infections during that week. The most common reasons respondents  gave for not providing resources or advice as frequently as they prescribed, administered or dispensed antibiotics were because resources were not available (18%), insufficient time (14%), or the patient was disinterested (12%).Sixty percent of respondents  stated that they had received information on avoiding unnecessary prescribing, administering or dispensing of antibiotics in the previous 12 months. Those who had received information received it most frequently at the work place (47%), through published guidelines (45%) or during group training (39%), and felt that the information had contributed to changing their view (58%), or had changed their practice (42%). The majority of those who did not change their practice based on the information received said this was because they were already following the principles of the message (82%), had no control over changing their practice (7%), found the information to be irrelevant to their current practice (5%), or had not had the opportunity (3%). Fifty-five percent of respondents  said that they would like to have more information on antibiotic resistance, while 41%  stated that prudent antibiotic use and information on antibiotic resistance had been well promoted during national campaigns in their country . Fewer than half the respondents across the EU/EEA countries had heard of European Antibiotic Awareness Day (EAAD),  or World Antimicrobial Awareness Week (WAAW) . Overall, for those who had heard of EAAD and WAAW, the majority were ‘undecided’  on their effectiveness in raising antibiotic awareness in their country, and 27% and 21%, respectively, believed EAAD and WAAW had been effective or very effective in raising antibiotic awareness in their country. Perceived effectiveness of the campaigns in raising awareness was found to vary across countries .Just over one third of respondents  stated they were prescribers of antibiotics, of whom 35% prescribed antibiotics daily. Medical doctors (80%) were the largest prescribing group, followed by dentists (12%) nurses (4%) and pharmacists (2%). Most prescribers  agreed that they had a key role in helping control antibiotic resistance, and 90%  also said they considered antibiotic resistance when treating a patient. A lower proportion (77%) agreed that they were confident in making antibiotic prescribing decisions, and while most respondents  agreed that they had easy access to the antibiotic guidelines they needed, only 70%  said they were confident in the antibiotic guidelines available to them.Thirty-one percent of prescribers said they would have preferred not to prescribe an antibiotic at least once in the week before completing the survey, but did so anyway. The most common reason for this was fear of patient deterioration or complications, with prescribers reporting that this fear affected their prescribing decision at least once per week (43%), or at least once per day (11%) . This reThis first EU/EEA-wide survey investigated healthcare workers’ knowledge of antibiotics, antibiotic use and antibiotic resistance, and whether they carry out the recommended behaviours on antibiotic use. Its results show a variation in healthcare workers’ responses across EU/EEA countries, and provide baseline evidence that may be useful for the development and evaluation of future interventions, both in individual countries and more broadly across the EU/EEA.The study exceeded the calculated required quota sample size, and there was also good representation from all the core healthcare professional groups who prescribe, administer or dispense antibiotics. This indicates that the findings are broadly representative of the relevant healthcare worker categories across the EU/EEA. Two limitations to consider are that those who responded to the survey may have an interest in tackling antimicrobial resistance (AMR) and 40% of respondents were medical doctors. However, it is worth noting that the majority of respondents do not have a formal role in tackling AMR, and all settings were represented. Another limitation was that while the estimated required quota was met/exceeded for the majority of countries, some countries had a higher representation from a particular healthcare professional group than others. There was also a higher response rate from women, although this is unsurprising since by 2016, 15 of the EU countries reported a higher number of female physicians . In addition, a substantial proportion of the nursing profession is female . Also, eThe findings of the survey highlight the need to continue to raise awareness about prudent use of antibiotics and antibiotic resistance, and also to enhance healthcare workers’ engagement in addressing these issues. They also highlight the need to design interventions based on education, resources and guidelines, which focus specifically on promoting behaviour that leads to prudent prescription, dispensing, and administration of antibiotics.As found in previous studies (predominantly focused on physicians), knowledge and perceived knowledge about antibiotics, antibiotic use and antibiotic resistance was high among healthcare workers, with more than 80% of respondents acknowledging the connection between prescribing, dispensing and administering antibiotics and the emergence and spread of antibiotic resistance -13,20,21Although more than 80% of respondents across all healthcare worker categories correctly answered the questions on the use of antibiotics, a much lower proportion were able to correctly answer questions related to the development and spread of antibiotic resistance. There was also a wide variation across professions in the proportion of respondents answering all seven knowledge questions correctly. The groups with the lowest knowledge and the groups who perceived they did not have sufficient knowledge on how to use antibiotics appropriately for their current practice should be targeted by future educational campaigns.It is reassuring that the survey questions relating to key and consistent messages promoted as part of previous EAAD campaigns throughout Europe i.e. that antibiotics are not effective against viruses, colds and flu, had the highest proportion of correct answers, and also that healthcare workers answered these questions more accurately than the general public in the Eurobarometer studies -7. HowevIncreasing engagement and promoting a sense of shared responsibility to tackle antibiotic resistance at individual, population and government levels, are potentially important means of bringing about behaviour change. One method that has previously been used is to focus on setting implementation intentions by pledging to take concrete action. This can be accomplished through, for example, joining a collective movement to cause change. Implementation intention is a method of encouraging individuals to decide in advance when, where and how they will act in order to reach a particular goal or objective. This approach uses what is described as if-then planning: if X happens, then I will do Y. For example, one of the pledges for general (primary care) practitioners used in the United Kingdom’s (UK) led Antibiotic Guardian campaign to support the UK Antimicrobial Resistance strategy states: ‘The next time I intend to prescribe antibiotics for a self-limiting infection to a patient with high expectations of antibiotic treatment, I will use a delayed/backup prescription’.The use of implementation intentions has been shown through meta-analyses to support both individuals and groups in bridging their intention-behaviour gaps ,26. EvalMost healthcare workers in our study who had direct patient or public involvement reported that they prescribed, dispensed or administered antibiotics at least once per week. While the vast majority of these interactions did not involve providing any written or oral advice, the most common barriers stated for not providing written resources to patients were that no resources were available, they had insufficient time, or the patient was not interested. It is therefore important that healthcare workers have access to appealing educational resources about antibiotics and antibiotic resistance when prescribing, dispensing or administering antibiotics. Although some materials about the importance of using antibiotics appropriately are available for the general public, these are often concerned with communicating the risks of antibiotic resistance or the need for prudent use of antibiotics in general terms. They are not patient-specific materials which provide information on, for example, the importance of taking antibiotics exactly as prescribed (dose/duration), and not to save them for later. Patient brochures covering topics such as ‘When should I worry?‘ , and ‘TrOur findings also point to the importance of ongoing training to enhance communication skills for those with direct patient contact. A cluster randomised control trial including primary care practices in five European countries representing north, south and central Europe , has previously shown that Internet-based training to enhance the communication skills of prescribers, including the use of a patient information booklet, achieved reductions in antibiotic prescription for respiratory tract infections across language and cultural boundaries . As thisRegarding the motivation for prescribing antibiotics, the findings highlight that clinicians‘ fear of serious bacterial infection and attempts to safeguard against the deterioration of a patient‘s health are important factors in their inappropriate prescribing of antibiotics. This points to the importance of developing rapid diagnostic tests/point-of-care tests that would remove this uncertainty, and thereby allow for more appropriate prescribing. Parallel media campaigns, which inform the public that they should trust their healthcare professional if they decide that antibiotics are not necessary for them could also be developed. Whatever interventions are implemented, it is important to evaluate their effectiveness, thereby ensuring a process of continual improvement. Countries could use the data from this study as a baseline for such evaluations, and use the survey tool as a means of assessing changes in the measured variables. However, it is important that the appropriate sample size for each country is determined at a national level.At EU/EEA level, it may be beneficial to consider developing a data repository platform to which countries can submit the results of their locally deployed survey findings, thereby facilitating benchmarking and monitoring at European level. In addition, this healthcare worker survey could be run at an EU/EEA-wide level at regular intervals or by individual countries in a similar manner to the Eurobarometer survey that focuses on the general public -7,While it is important to continue EU-level awareness campaigns such as the EAAD, interventions at national and local level are key to changing antibiotic-related behaviours of healthcare workers. Previous systematic reviews have shown that the effectiveness of an intervention on antibiotic prescribing depends to a large extent on the particular prescribing behaviour and any barriers to change that may exist within the targeted community. In addition, multi-faceted educational interventions occurring on multiple levels are only effective after addressing such local barriers to change ,24,35. EWhile several studies in Europe have assessed antibiotic-related knowledge and attitudes of members of the public, healthcare students or individual professional groups, there is a paucity of equivalent literature that focuses on healthcare workers. To our knowledge, this is the first multi-country and multi-professional study on the knowledge, attitudes and behaviours of healthcare workers regarding antibiotics, antibiotic use and antibiotic resistance, and it has identified important knowledge gaps such as ‘Every person treated with antibiotics is at an increased risk of antibiotic resistant infection’, ‘Antibiotic resistant bacteria can spread from person to person’, and ‘Healthy people can carry antibiotic resistant bacteria’ that need to be addressed. While some of these can be addressed at EU/EEA level, individual countries should review the data presented in this study, and use these to develop a tailored approach for their own context.